Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of Sendai virus antigen peptide composition and infects the application in detecting Sendai virus, Sendai virus antigen peptide composition provided by the invention has improved specificity and the specific aim of polypeptides in combination, therefore can improve the accuracy of laboratory animal microorganism detection.
The invention provides a kind of Sendai virus antigen peptide composition, comprise there is the polypeptide of sequence as shown in SEQ ID NO:1, as shown in SEQ ID NO:2 sequence polypeptide and as shown in SEQ ID NO:3 the polypeptide of sequence.
The present invention also provides a kind of Sendai virus antigen peptide composition, is included in to have as shown in SEQ ID NO:1 in sequence the polypeptide that replaces, lack or add one or more amino-acid residues and obtain, replace, lack or add the polypeptide that one or more amino-acid residues obtain having as shown in SEQ ID NO:2 to replace, lack or add the polypeptide that one or more amino-acid residues obtain in sequence and have as shown in SEQ ID NO:3 in sequence.
Preferably, in Sendai virus antigen peptide composition provided by the invention, replace, lack or add the aminoacid sequence of polypeptide and the homology of the aminoacid sequence shown in SEQ ID NO:1 that one or more amino-acid residues obtain and be greater than 85% having as shown in SEQ ID NO:1 in sequence;
Preferably, in Sendai virus antigen peptide composition provided by the invention, replace, lack or add the aminoacid sequence of polypeptide and the homology of the aminoacid sequence shown in SEQ ID NO:2 that one or more amino-acid residues obtain and be greater than 85% having as shown in SEQ ID NO:2 in sequence;
Preferably, in Sendai virus antigen peptide composition provided by the invention, replace, lack or add the aminoacid sequence of polypeptide and the homology of the aminoacid sequence shown in SEQ ID NO:3 that one or more amino-acid residues obtain and be greater than 85% having as shown in SEQ ID NO:3 in sequence.
Antigen peptide in antigen peptide composition provided by the invention is the dominant antigen epi-position of Sendai virus, and molecular weight, therefore reduced the possibility with other molecule generation cross reactions, improved specificity and the specific aim of polypeptides in combination, thereby improved, Sendai virus has been infected to the accuracy rate detecting.
Preferably, the DNA molecular of coding aminoacid sequence as shown in SEQ ID NO:1, has the nucleotide sequence as shown in SEQ ID NO:4.
Preferably, the DNA molecular of coding aminoacid sequence as shown in SEQ ID NO:2, has the nucleotide sequence as shown in SEQ ID NO:5.
Preferably, the DNA molecular of coding aminoacid sequence as shown in SEQ ID NO:3, has the nucleotide sequence as shown in SEQ ID NO:6.
The present invention also provides a kind of Sendai virus vaccine, comprises Sendai virus antigen peptide composition provided by the invention and pharmaceutically acceptable auxiliary material.
Wherein, Sendai virus antigen peptide composition provided by the invention, comprise the polypeptide with sequence as shown in SEQ ID NO:1, the polypeptide of sequence shown in the polypeptide of sequence shown in SEQ ID NO:2 and SEQ ID NO:3, or be included in to have as shown in SEQ ID NO:1 and replace in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or add the polypeptide that one or more amino-acid residues obtain.
Preferably, the formulation of Sendai virus vaccine provided by the invention is oral preparations or injection.
Because the antigen peptide in antigen peptide composition provided by the invention is the dominant antigen epi-position of Sendai virus, so when preparing vaccine, can reduce the harm of Sendai virus to animal.
In addition, the present invention also provides Sendai virus antigen peptide composition provided by the invention to infect the application in detecting Sendai virus.
Wherein, Sendai virus antigen peptide composition provided by the invention, comprise the polypeptide with sequence as shown in SEQ ID NO:1, the polypeptide of sequence shown in the polypeptide of sequence shown in SEQ ID NO:2 and SEQ ID NO:3, or be included in to have as shown in SEQ ID NO:1 and replace in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or add the polypeptide that one or more amino-acid residues obtain.
With the Dominant Epitopes of antigen, detecting the principle whether animal body infect this antigen is: when animal body infects after antigen, animal body will produce the antibody for this antigen, this antibody is distributed in the serum of animal, existence by detecting this antibody in serum whether, just can learn whether animal body infects antigen.The antibody that animal body produces is generally the Dominant Epitopes for antigen, and the antibody capable that produced is specific to combine with the Dominant Epitopes of antigen.So detect the antibody whether existing in serum with its combination by antigen Dominant Epitopes, just can learn whether animal body infects this virus.
The invention provides a kind of non-diagnostic purpose and detect the method that Sendai virus infects, comprise the following steps:
Step 1: antigen peptide composition provided by the invention is coated with on enzyme plate, through the first washing rear enclosed, obtains the enzyme plate after sealing;
Step 2: get testing sample and add in the enzyme plate after sealing, the colour developing through hatching, after anti-, the 3rd washing of the second washing, mark two, obtains and examined solution;
Step 3: get and examined solution, detect OD by microplate reader 450value, adopts Cut off value method judgement detected result;
The standard of Cut off value method judgement is:
Examined the OD of solution
450value
it is positive,
Examined the OD of solution
450value
it is negative,
examined the OD of solution
450value
for suspicious, again survey the OD that is examined solution
450be worth, examined the OD of solution
450value is less than
be judged to feminine gender;
Wherein,
oD for all negative control samples
450mean value, SD is all negative sample OD
450standard deviation; Negative control sample is consistent with the detection method of testing sample;
Wherein, Sendai virus antigen peptide composition provided by the invention, comprise the polypeptide with sequence as shown in SEQ ID NO:1, the polypeptide of sequence shown in the polypeptide of sequence shown in SEQ ID NO:2 and SEQ ID NO:3, or be included in to have as shown in SEQ ID NO:1 and replace in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or add the polypeptide that one or more amino-acid residues obtain.
value and SD value are fixed value for a certain species, can after detection, preserve, and while again detecting, can negative control not detected.
As preferably, be coated with and be specially: the coated damping fluid that 100 μ L are contained to 10 μ g/mL antigen peptide compositions adds on enzyme plate, under 4 â of conditions, places and spends the night.
Preferably, the pH of coated damping fluid is 9.6.
More preferably, the solvent of coated damping fluid is distilled water.
Preferred, Na in coated damping fluid 2cO 3quality-volumetric concentration be 1.59g/L, NaHCO 3mass body volume concentrations be 2.93g/L.
As preferably, the first washing adopts lavation buffer solution.
Preferably, lavation buffer solution lavation buffer solution pH value is 7.4.
More preferably, the solvent of lavation buffer solution is distilled water.
More preferably, in lavation buffer solution, the content of each component is:
As preferably, sealing is specially: on enzyme plate, add sample diluting liquid, under 37 â of conditions, place and be no less than 1 hour.
Preferably, sample diluting liquid is the mixture of bovine serum albumin and lavation buffer solution.
More preferably, in sample diluting liquid, the content of bovine serum albumin is 1g/L.
As preferably, testing sample is the mixture of animal serum and sample diluting liquid.
Preferably, in testing sample, the volume ratio of animal serum and sample diluting liquid is 1:2.
As preferably, the addition of testing sample is, every hole 50 μ L.
As preferably, hatch and be specially: at ambient temperature, place 1 hour.
As preferably, the second washing adopts lavation buffer solution.
As preferably, two anti-employing horseradish peroxidase-labeled.
Preferably, the dilution two of employing sample diluting liquid is anti-, and two after preparation dilution resists.
More preferably, two volume ratios anti-and sample diluting liquid are 1:10000.
Most preferably, mark two is anti-to be specially: get two after 100 μ L dilutions and anti-ly add enzyme plate, under 37 â of conditions, place 1 hour.
As preferably, the 3rd washing adopts lavation buffer solution.
As preferably, colour developing is specially: on enzyme plate, add substrate buffer solution, place after 5min, add stop buffer.
Preferably, the pH value of substrate buffer solution is 5.0.
More preferably, the solvent of substrate buffer solution be water, wherein the content of each component is:
Na 2HPO 4 0.1028mol/L
Citric acid 0.0486mol/L
More preferably, stop buffer adopts the H that concentration is 2mol/L 2sO 4the aqueous solution.
The invention provides a kind of Sendai virus and infect detection kit, comprise the flat board, lavation buffer solution, sample diluting liquid, stop buffer and the substrate buffer solution that are coated with antigen peptide composition provided by the invention.
Wherein, Sendai virus antigen peptide composition provided by the invention, comprise the polypeptide with sequence as shown in SEQ ID NO:1, the polypeptide of sequence shown in the polypeptide of sequence shown in SEQ ID NO:2 and SEQ ID NO:3, or be included in to have as shown in SEQ ID NO:1 and replace in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or add the polypeptide that one or more amino-acid residues obtain.
Preferably, the dull and stereotyped matrix that is coated with antigen peptide composition provided by the invention is: slide, enzyme plate, polypeptide chip or protein chip.
More preferably, the dull and stereotyped matrix that is coated with antigen peptide composition provided by the invention is: polypeptide chip.
Preferably, lavation buffer solution lavation buffer solution pH value is 7.4.
More preferably, the solvent of lavation buffer solution is distilled water.
Preferably, in lavation buffer solution, the content of each component is:
Preferably, sample diluting liquid is the mixture of bovine serum albumin and lavation buffer solution.
More preferably, in sample diluting liquid, the content of bovine serum albumin is 1g/L.
Preferably, stop buffer is that concentration is the H of 2mol/L 2sO 4the aqueous solution.
Preferably, the pH value of substrate buffer solution is 5.0.
More preferably, the solvent of substrate buffer solution is water.
More preferably, in substrate buffer solution, the content of each component is:
Na 2HPO 4 0.1028mol/L
Citric acid 0.0486mol/L
As preferably, the using method that Sendai virus provided by the invention infects detection kit is:
Step 1: add in the flat board that is coated with antigen peptide composition provided by the invention after sample thief diluted testing sample, the colour developing through hatching, after anti-, the 5th washing of the 4th washing, mark two, obtains and examined solution;
Step 2: get and examined solution, detect OD by microplate reader 450value, adopts Cut off value method judgement detected result;
The standard of Cut off value method judgement is:
Examined the OD of solution
450value
it is positive,
Examined the OD of solution
450value
it is negative,
examined the OD of solution
450value
for suspicious, again survey the OD that is examined solution
450be worth, examined the OD of solution
450value is less than
be judged to feminine gender;
Wherein,
oD for all negative control samples
450mean value, SD is all negative sample OD
450standard deviation, negative control sample is consistent with the detection method of testing sample.
value and SD value are fixed value for a certain species, can after detection, preserve, and while again detecting, can negative control not detected.
Preferably, in testing sample, the volume ratio of animal serum and sample diluting liquid is 1:2.
As preferably, the addition of testing sample is, every hole 50 μ L.
As preferably, hatch and be specially: at ambient temperature, place 1 hour.
As preferably, the second washing adopts lavation buffer solution.
As preferably, two anti-employing horseradish peroxidase-labeled.
Preferably, the dilution two of employing sample diluting liquid is anti-, and two after preparation dilution resists.
More preferably, two volume ratios anti-and sample diluting liquid are 1:10000.
Most preferably, mark two is anti-to be specially: get two after 100 μ L dilutions and anti-ly add enzyme plate, under 37 â of conditions, place 1 hour.
As preferably, the 3rd washing adopts lavation buffer solution.
As preferably, colour developing is specially: on enzyme plate, add substrate buffer solution, place after 5min, add stop buffer.
The invention provides a kind of Sendai virus antigen peptide composition, comprise the polypeptide with sequence as shown in SEQ ID NO:1, the polypeptide of sequence shown in the polypeptide of sequence shown in SEQ ID NO:2 and SEQ ID NO:3, or be included in to have as shown in SEQ ID NO:1 and replace in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or add the polypeptide that one or more amino-acid residues obtain.And provide it Sendai virus, to infect the application in detecting.The present invention adopts protein hybridization technology, filters out the dominant antigen of Sendai virus, the NP(nucleocapsid protein that this dominant antigen is Sendai virus) albumen, the NP albumen of Sendai virus has 524 amino-acid residues.The present invention, according to the aminoacid sequence of dominant antigen NP albumen, adopts overlapping mode design antigenic peptide array and synthesized antigenic peptide array chip, has filtered out Sendai virus high specificity and the little linear epitope of molecular weight.Because the antigen peptide molecule amount in Sendai virus antigen peptide composition provided by the invention is less, reduced the possibility with other molecule generation cross reactions, therefore improve specificity and the specific aim of polypeptides in combination, thereby improved the accuracy of laboratory animal microorganism detection.The present invention also provides a kind of Sendai virus vaccine, comprise Sendai virus antigen peptide composition provided by the invention and pharmaceutically acceptable auxiliary material, because the anti-Yantai in antigen peptide composition provided by the invention is the dominant antigen of Sendai virus, so when preparing vaccine, can reduce the harm of Sendai virus to animal.Experiment shows: antigen peptide composition provided by the invention is compared with the full particulate antigen of Sendai virus, and antigen peptide composition provided by the invention is lower to the detected value of negative serum peptide combination, reflects peptide combination and has lower reaction background, and specificity is better.
Embodiment
The invention provides and a kind of Sendai virus antigen peptide composition is provided and infects the application in detecting Sendai virus, those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications as herein described or suitably change and combination within not departing from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
The invention provides a kind of Sendai virus antigen peptide composition, comprise the polypeptide of sequence shown in the polypeptide of sequence shown in the polypeptide, SEQ ID NO:2 with sequence as shown in SEQ ID NO:1 and SEQ ID NO:3.
The present invention also provides a kind of Sendai virus antigen peptide composition, is included in to have as shown in SEQ ID NO:1 in sequence the polypeptide that replaces, lack or add one or more amino-acid residues and obtain, replace, lack or add the polypeptide that one or more amino-acid residues obtain having as shown in SEQ ID NO:2 to replace, lack or add the polypeptide that one or more amino-acid residues obtain in sequence and have as shown in SEQ ID NO:3 in sequence.
In Sendai virus antigen peptide composition provided by the invention, replace, lack or add the aminoacid sequence of polypeptide and the homology of the aminoacid sequence shown in SEQ ID NO:1 that one or more amino-acid residues obtain and be greater than 85% having as shown in SEQ ID NO:1 in sequence;
In Sendai virus antigen peptide composition provided by the invention, replace, lack or add the aminoacid sequence of polypeptide and the homology of the aminoacid sequence shown in SEQ ID NO:2 that one or more amino-acid residues obtain and be greater than 85% having as shown in SEQ ID NO:2 in sequence;
In Sendai virus antigen peptide composition provided by the invention, replace, lack or add the aminoacid sequence of polypeptide and the homology of the aminoacid sequence shown in SEQ ID NO:3 that one or more amino-acid residues obtain and be greater than 85% having as shown in SEQ ID NO:3 in sequence.
In Sendai virus antigen peptide composition provided by the invention, the content of each component is not limit.
Antigen peptide in antigen peptide composition provided by the invention is the dominant antigen of Sendai virus, and molecular weight, therefore reduced with other molecule generation cross reactions may, improved specificity and the specific aim of polypeptides in combination, thereby improved, Sendai virus is infected to the accuracy rate detecting.
In antigen peptide composition provided by the invention, the screening method of antigen peptide is: first, adopt protein hybridization technology, filter out the dominant antigen of Sendai virus; Then, according to the aminoacid sequence of dominant antigen, adopt overlapping mode design antigenic peptide array and synthesized antigenic peptide array chip, filtered out Sendai virus linear epitope.
The Sendai virus dominant antigen filtering out of the present invention is the NP(nucleocapsid protein of Sendai virus) albumen.
During design antigenic peptide array, take 14ï½18 amino-acid residues as polypeptide length, with 2 amino acid displacements, design antigenic peptide array.
The automatic single-point Peptide synthesizer of synthetic employing of antigen peptide array.
The dominant antigen peptide epitopes filtering out is positioned at the C end of NP albumen.
Antigen peptide in antigen peptide composition provided by the invention is the dominant antigen epi-position of Sendai virus, and molecular weight, therefore reduced the possibility with other molecule generation cross reactions, improved specificity and the specific aim of polypeptides in combination, thereby improved, Sendai virus has been infected to the accuracy rate detecting.
The preparation of the antigen peptide in antigen peptide composition provided by the invention can adopt biological method or chemosynthesis.
The DNA molecular of coding aminoacid sequence as shown in SEQ ID NO:1, has the nucleotide sequence as shown in SEQ ID NO:4.
The DNA molecular of coding aminoacid sequence as shown in SEQ ID NO:2, has the nucleotide sequence as shown in SEQ ID NO:5.
The DNA molecular of coding aminoacid sequence as shown in SEQ ID NO:3, has the nucleotide sequence as shown in SEQ ID NO:6.
The present invention also provides a kind of Sendai virus vaccine, comprises Sendai virus antigen peptide composition provided by the invention and pharmaceutically acceptable auxiliary material.
Wherein, Sendai virus antigen peptide composition provided by the invention, comprise the polypeptide with sequence as shown in SEQ ID NO:1, the polypeptide of sequence shown in the polypeptide of sequence shown in SEQ ID NO:2 and SEQ ID NO:3, or be included in to have as shown in SEQ ID NO:1 and replace in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or add the polypeptide that one or more amino-acid residues obtain.
For convenience of using, the invention provides to the formulation of Sendai virus vaccine be oral preparations or injection.
Because the antigen peptide in antigen peptide composition provided by the invention is the dominant antigen epi-position of Sendai virus, so when preparing vaccine, can reduce the harm of Sendai virus to animal.
Sendai virus antigen peptide composition provided by the invention infects the application in detecting Sendai virus.
Sendai virus antigen peptide composition provided by the invention, comprise the polypeptide with sequence as shown in SEQ ID NO:1, the polypeptide of sequence shown in the polypeptide of sequence shown in SEQ ID NO:2 and SEQ ID NO:3, or be included in to have as shown in SEQ ID NO:1 and replace in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or add the polypeptide that one or more amino-acid residues obtain.
With the Dominant Epitopes of antigen, detecting the principle whether animal body infect this antigen is: when animal body infects after antigen, animal body will produce the antibody for this antigen, this antibody is distributed in the serum of animal, existence by detecting this antibody in serum whether, just can learn whether animal body infects antigen.The antibody that animal body produces is generally the Dominant Epitopes for antigen, and the antibody capable that produced is specific to combine with the Dominant Epitopes of antigen.So detect the antibody whether existing in serum with its combination by antigen Dominant Epitopes, just can learn whether animal body infects this virus.
The invention provides a kind of non-diagnostic purpose and detect the method that Sendai virus infects, comprise the following steps: first, antigen peptide composition provided by the invention is coated with on enzyme plate, through the first washing rear enclosed, obtains the enzyme plate after sealing; Then, get testing sample and add in the enzyme plate after sealing, the colour developing through hatching, after anti-, the 3rd washing of the second washing, mark two, obtains and is examined solution; Then, get and examined solution, by microplate reader, detect OD 450value, adopts Cut off value method judgement detected result;
The standard of Cut off value method judgement is:
Examined the OD of solution
450value
it is positive,
Examined the OD of solution
450value
it is negative,
examined the OD of solution
450value
for suspicious, again survey the OD that is examined solution
450be worth, examined the OD of solution
450value is less than
be judged to feminine gender;
Wherein,
oD for all negative control samples
450mean value, SD is all negative sample OD
450standard deviation; Negative control sample is consistent with the detection method of testing sample;
Wherein, Sendai virus antigen peptide composition provided by the invention, comprise the polypeptide with sequence as shown in SEQ ID NO:1, the polypeptide of sequence shown in the polypeptide of sequence shown in SEQ ID NO:2 and SEQ ID NO:3, or be included in to have as shown in SEQ ID NO:1 and replace in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or add the polypeptide that one or more amino-acid residues obtain.
value and SD value are fixed value for a certain species, can after detection, preserve, and while again detecting, can negative control not detected.
Non-diagnostic purpose provided by the invention detects in the method for Sendai virus infection, coated being specially: the coated damping fluid that 100 μ L are contained to 10 μ g/mL antigen peptide compositions adds on enzyme plate, places and spends the night under 4 â of conditions.
The pH that wraps adopted coated damping fluid is 9.6.
The solvent of coated damping fluid is distilled water.
Na in coated damping fluid 2cO 3quality-volumetric concentration be 1.59g/L, NaHCO 3mass body volume concentrations be 2.93g/L.
In the method that the detection Sendai virus of non-diagnostic purpose provided by the invention infects, the first washing adopts lavation buffer solution.
Lavation buffer solution lavation buffer solution pH value is 7.4.
The solvent of lavation buffer solution is distilled water.
In lavation buffer solution, the content of each component is:
Non-diagnostic purpose provided by the invention detects in the method for Sendai virus infection, and sealing is specially: on enzyme plate, add sample diluting liquid, under 37 â of conditions, place and be no less than 1 hour.
Sample diluting liquid is the mixture of bovine serum albumin and lavation buffer solution.
In sample diluting liquid, the content of bovine serum albumin is 1g/L.
Non-diagnostic purpose provided by the invention detects in the method for Sendai virus infection, and testing sample is the mixture of animal serum and sample diluting liquid.
In testing sample, the volume ratio of animal serum and sample diluting liquid is 1:2.
The addition of testing sample is every hole 50 μ L.
Non-diagnostic purpose provided by the invention detects in the method for Sendai virus infection, hatches and is specially: at ambient temperature, place 1 hour.
Non-diagnostic purpose provided by the invention detects in the method for Sendai virus infection, and the second washing adopts lavation buffer solution.
Non-diagnostic purpose provided by the invention detects in the method for Sendai virus infection, two anti-employing horseradish peroxidase-labeled.
The dilution two of employing sample diluting liquid is anti-, and two after preparation dilution resists.
Two volume ratios anti-and sample diluting liquid are 1:10000.
Mark two is anti-to be specially: get two after 100 μ L dilutions and anti-ly add enzyme plate, under 37 â of conditions, place 1 hour.
Non-diagnostic purpose provided by the invention detects in the method for Sendai virus infection, and the 3rd washing adopts lavation buffer solution.
Non-diagnostic purpose provided by the invention detects in the method for Sendai virus infection, and colour developing is specially: on enzyme plate, add substrate buffer solution, place after 5min, add stop buffer.
The pH value of substrate buffer solution is 5.0.
The solvent of substrate buffer solution be water, wherein the content of each component is:
Na 2HPO 4 0.1028mol/L
Citric acid 0.0486mol/L
Stop buffer adopts the H that concentration is 2mol/L 2sO 4the aqueous solution.
The invention provides a kind of Sendai virus and infect detection kit, comprise the flat board, lavation buffer solution, sample diluting liquid, stop buffer and the substrate buffer solution that are coated with antigen peptide composition provided by the invention.
Sendai virus antigen peptide composition provided by the invention, comprise the polypeptide with sequence as shown in SEQ ID NO:1, the polypeptide of sequence shown in the polypeptide of sequence shown in SEQ ID NO:2 and SEQ ID NO:3, or be included in to have as shown in SEQ ID NO:1 and replace in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or add the polypeptide that one or more amino-acid residues obtain.
Being coated with the dull and stereotyped matrix that is coated with antigen peptide composition provided by the invention provided by the invention is: slide, enzyme plate, polypeptide chip or protein chip.
The dull and stereotyped matrix that is coated with antigen peptide composition provided by the invention is: polypeptide chip.
Lavation buffer solution lavation buffer solution pH value is 7.4.
The solvent of lavation buffer solution is distilled water.
In lavation buffer solution, the content of each component is:
Sample diluting liquid is the mixture of bovine serum albumin and lavation buffer solution.
In sample diluting liquid, the content of bovine serum albumin is 1g/L.
Stop buffer is that concentration is the H of 2mol/L 2sO 4the aqueous solution.
The pH value of substrate buffer solution is 5.0.
The solvent of substrate buffer solution be water.
In substrate buffer solution, the content of each component is:
Na 2HPO 4 0.1028mol/L
Citric acid 0.0486mol/L
The using method that Sendai virus provided by the invention infects detection kit is: first, after sample thief diluted testing sample, add in the flat board that is coated with antigen peptide composition provided by the invention, the colour developing through hatching, after anti-, the 5th washing of the 4th washing, mark two, obtains and is examined solution; Then, get and examined solution, by microplate reader, detect OD 450value, adopts Cut off value method judgement detected result;
The standard of Cut off value method judgement is:
Examined the OD of solution
450value
it is positive,
Examined the OD of solution
450value
it is negative,
examined the OD of solution
450value
for suspicious, again survey the OD that is examined solution
450be worth, examined the OD of solution
450value is less than
be judged to feminine gender;
Wherein,
oD for all negative control samples
450mean value, SD is all negative sample OD
450standard deviation; Negative control sample is consistent with the detection method of testing sample.
value and SD value are fixed value for a certain species, can after detection, preserve, and while again detecting, can negative control not detected.
Sendai virus provided by the invention infects in the using method of detection kit, and in testing sample, the volume ratio of animal serum and sample diluting liquid is 1:2.
The addition of testing sample is, every hole 50 μ L.
Hatch and be specially: at ambient temperature, place 1 hour.
The second washing adopts lavation buffer solution.
Two anti-employing horseradish peroxidase-labeled.
The dilution two of employing sample diluting liquid is anti-, and two after preparation dilution resists.
Two volume ratios anti-and sample diluting liquid are 1:10000.
Mark two is anti-to be specially: get two after 100 μ L dilutions and anti-ly add enzyme plate, under 37 â of conditions, place 1 hour.
The 3rd washing adopts lavation buffer solution.
Colour developing is specially: on enzyme plate, add substrate buffer solution, place after 5min, add stop buffer.
The invention provides a kind of Sendai virus antigen peptide composition, comprise the polypeptide with sequence as shown in SEQ ID NO:1, the polypeptide of sequence shown in the polypeptide of sequence shown in SEQ ID NO:2 and SEQ ID NO:3, or be included in to have as shown in SEQ ID NO:1 and replace in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or add the polypeptide that one or more amino-acid residues obtain.And provide it Sendai virus, to infect the application in detecting.The present invention adopts protein hybridization technology, filters out the dominant antigen of Sendai virus, the NP(nucleocapsid protein that this dominant antigen is Sendai virus) albumen, the NP albumen of Sendai virus has 524 amino-acid residues.The present invention, according to the aminoacid sequence of dominant antigen NP albumen, adopts overlapping mode design antigenic peptide array and synthesized antigenic peptide array chip, has filtered out Sendai virus high specificity and the little linear epitope of molecular weight.Because the antigen peptide molecule amount in Sendai virus antigen peptide composition provided by the invention is less, reduced the possibility with other molecule generation cross reactions, therefore improve specificity and the specific aim of polypeptides in combination, thereby improved the accuracy of laboratory animal microorganism detection.The present invention also provides a kind of Sendai virus vaccine, comprise Sendai virus antigen peptide composition provided by the invention and pharmaceutically acceptable auxiliary material, due to the antigen peptide composition provided by the invention dominant antigen that is Sendai virus, so when preparing vaccine, can reduce the harm of Sendai virus to animal.Experiment shows: antigen peptide composition provided by the invention is compared with the full particulate antigen of Sendai virus, and antigen peptide composition provided by the invention is lower to the detected value of negative serum peptide combination, reflects peptide combination and has lower reaction background, and specificity is better.
The examination material that the present invention adopts is all common commercially available product, all can be buied by market.
Below in conjunction with embodiment, further set forth the present invention:
Determining of the dominant antigen albumen of embodiment 1 Sendai virus
First, preparation SDS-PAGE electrophoresis solution used:
1.0mol/L Tris(pH6.8) solution: get 12g Tris and be dissolved in 80mL distilled water, adjust pH to 6.8, is settled to 100mL with distilled water;
1.5mol/L Tris(pH8.8) solution: get 18g Tris and be dissolved in 80mL distilled water, adjust pH to 8.8, is settled to 100mL with distilled water;
10%SDS solution: get 10g SDS and be dissolved in 80mL distilled water, regulate pH value to 7.2, be settled to 100mL with distilled water;
10% ammonium persulfate solution: get 1g Ammonium Persulfate 98.5 and be dissolved in 10mL distilled water;
5 * Tris-glycine electrophoretic buffer: Tris15.1g, glycine 94g, adds SDS5g after being dissolved in 800mL distilled water completely, is settled to 1L, before use, be diluted to 1 *;
30% acrylamide stock solution: get 1g methylene-bisacrylamide and be dissolved in 80mL water, heating for dissolving.Then add 29g acrylamide stirring and dissolving, with distilled water, be settled to 100mL;
5 * sample-loading buffer: get 1mol/L Tris-Cl(pH6.8) solution 1.25mL, 0.5g SDS, 25mg tetrabromophenol sulfonphthalein, 2.5mL glycerine, adds deionized water dissolving to be settled to 5mL, adds 250 μ L beta-mercaptoethanols or 2.5mL1mol/L DTT before use;
The method of determining the dominant antigen albumen of Sendai virus is:
The sample-loading buffer of getting Sendai virus antigen 80 μ L and 20 μ L mixes, and boils to be splined on polyacrylamide gel after 5minï½10min and to carry out SDS-PAGE electrophoresis in boiling water, and sample adopts 60V voltage when basic unit's glue, adopts 120V voltage during to separation gel.After sample-loading buffer is run out of, according to Marker, near glue object band is cut.According to placing sample, 60V voltage under transfer printing 3h by glue to the order of film (nitrocellulose filter) to white (transfer printing folder) by black.Nitrocellulose filter is sealed to 1h by 5% skim-milk room temperature.The rat blood serum of getting respectively normal rat serum and infection Sendai virus dilutes with 1:500 with 5% skim-milk, in 4 â of overnight incubation.Second day is washed film three times with TBS-T solution, each 10min.Get two anti-(sheep anti mouse) IgG-HRP, with 5% skim-milk, with 1:2000, dilute, room temperature and film are hatched 1h.With TBS-T solution, wash film three times, each 10min.Add and strengthen luminescent solution at scotography.
The rat blood serum of normal rat serum and infection Sendai virus and Sendai virus antigen results of hybridization are as shown in Figure 1.Arrow a indication band molecular weight is 70kDa, and arrow b indication band molecular weight is 57kDa.Show to only have the track of hatching with the rat blood serum that infects Sendai virus just to have hybridization development band, and the band content that the indicated molecular weight of arrow b is 57kDa is the highest.Due to Sendai virus NP(nucleocapsid protein) molecular weight of albumen is about 57kDa, and research shows in Sendai virus course of infection, animal body produces for the titre of the antibody of NP albumen very high, so the indicated band of arrow b is Sendai virus NP albumen.Above-mentioned experimental result shows that the dominant antigen of Sendai virus is Sendai virus NP albumen.
The screening of embodiment 2 Sendai virus dominant antigen polypeptide epitopes
According to 524 of NP albumen amino acid whose protein sequences, with polypeptide array technique, 2 polypeptide arrays have been made.Array chip designs in overlapping mode, and with 14 amino acid polypeptide length, 2 amino acid displacements are calculated, and arranges 264 polypeptide on every polypeptide array, and two array chips are totally 528 polypeptide.
Configure required solution:
Concentration is the Fmoc-amino acid solution of 0.25mol/L;
The dimethyl formamide solution of confining liquid I for containing 2% (v/v) diacetyl oxide;
Confining liquid II is the dimethyl formamide solution of DIPEA of the diacetyl oxide and 2% (v/v) of 2% (v/v);
Fmoc-removes the dimethyl formamide solution of liquid for containing 20% (v/v) piperidines.
First, synthesize polypeptide array:
The PEG-cellulose membrane of activation is placed on Autos potter, and the Fmoc-amino acid solution that automatically adds 0.1 μ L according to program reacts with film to the specific position on the PEG-cellulose membrane of activation, reacts 20 minutes, repeats this membrane process; PEG-cellulose membrane is first immersed in confining liquid I and reacts and in immersing confining liquid II, react 20 minutes for 5 minutes, carry out side chain sealing, then with DMF, clean 4 times.Then, add Fmoc-to remove in liquid PEG-cellulose membrane and react 5min, repeat 1 time, to remove N-terminal Fmoc blocking group; Go after blocking group, with DMF, clean 3 times, each 30 seconds, then dry with ethanol again; Repeat above step, until each polypeptide is all synthetic.After all synthesizing, first with TFA cocktail, remove side chain protected group, then use CH 2cl 2clean 4 times, then dry with ethanol.Product is in-20 â of preservations, for next step immune response experiment.
According to experimental design, polypeptide automatic DNA synthesizer DNA synthesizes polypeptide array, polypeptide array pattern figure as shown in Figure 2, every polypeptide array is laterally 11 polypeptide points, be longitudinally 24 polypeptide points, in figure, each polypeptide point on polypeptide chip has been carried out to Position Number mark, the corresponding corresponding peptide sequence of each numbering.Positive epi-position numeral in the interpretation of all polypeptide arrays is corresponding one by one with above-mentioned reference numbers and peptide sequence numbering below.
By two polypeptide array chips and two kinds of experimental rat serum, 1 part of negative control group rat blood serum, 1 part of positive group rat blood serum, has carried out respectively immune response detection, comprises the following steps:
Sealing: with containing massfraction, be that 4% skimmed milk and massfraction are the TBS-T damping fluid of 5% sucrose, room temperature reaction 4 hours; Primary antibodie is hatched: getting rat blood serum is 1:500 dilution by volume; React with polypeptide array, spend the night at 4 â; Wash film: with TBS-T damping fluid rinsing 3 times, each 10 minutes; Two anti-hatching: two anti-(sheep anti mouse) IgG-HRP, the volume ratio dilution of pressing 1:2000 with confining liquid, seals under room temperature 2 hours; Wash film: with TBS-T damping fluid rinsing 3 times, each 10 minutes; Colour developing: add ECL luminescence reagent, digital imagery.
Polypeptide array is combined result as shown in Figure 3 with rat blood serum, and wherein, Fig. 3 (a) is gained image (being derived from imager analysis software synthetic image) after control serum reacts with array, gained image after the positive serum of Fig. 3 (b) reacts with array.From the overall contrast of polypeptide array detection figure, in serum, positive reflecting point mainly concentrates on rear 1/3 position of chip, shows that the Main Antigenic of Sendai virus NP albumen mainly concentrates on the C end of this albumen.Can observe main four epitope regions with significant difference, by 1,2,3 and No. 4 circle, go out respectively simultaneously.By the comparison to colour developing point in above-mentioned two figure, in negative control sera for 229ï½232(2 frame), 241ï½243(3 frame) and 258ï½259(4 frame) almost not reaction of epitope, and positive serum is stronger to above-mentioned epi-position reaction, and be continuous a plurality of polypeptide point colour developing, showing that above-mentioned epi-position can excite stronger immune response, may be main epitope.The polypeptide epitope positive serum response intensity of No. 1 frame is apparently higher than negative serum, but it does not form continuous difference colour developing site, judges that it may be epitope, also needs further checking.For the antibody response of other epi-positions, in two groups, there is not obvious difference.For the non-specific colour developing point occurring, may be the exposure due to polypeptide antagonist recognition site, and the cross reaction of Serum Antibody cause.Due in negative control sera for 229ï½232(2 frame) almost not reaction of epitope, and positive serum is stronger to above-mentioned epi-position reaction, and be continuous a plurality of polypeptide point colour developing, show that above-mentioned epi-position can excite stronger immune response, so 4 polypeptide that the present invention chooses in No. 2 frames are done further research.
Embodiment 3 Sendai virus antigenic peptide composition Function Identification
Adopt the method for chemosynthesis to prepare 4 polypeptide that the embodiment of the present invention 2 filters out, difference called after antigen A, antigen B, antigens c, antigen D, and be coated with to ELISA Sptting plate with 1mg/mL concentration, to do and react with Sendai virus positive serum and negative serum respectively, result is as shown in Figure 4.Result shows the Sendai virus with natural antigen SeV() compare, a little less than 4 peptide based immunogens.But antigen A, antigens c and antigen D still show good immunogenicity, and antigen B antigenicity is poor.Wherein, the aminoacid sequence of antigen A is as shown in SEQ ID NO:1, and the aminoacid sequence of antigen B is as shown in SEQ ID NO:2, and the aminoacid sequence of antigens c is as shown in SEQ ID NO:3.
The full particulate antigen function ratio of embodiment 4 Sendai virus antigenic peptide provided by the invention composition and Sendai virus
Adopt the method for chemosynthesis to prepare 3 polypeptide that the embodiment of the
present invention3 filters out, be respectively antigen A, antigens c, antigen D, and be coated with to ELISA Sptting plate with 1mg/mL concentration, detect respectively 17 parts of mice serum samples, result as shown in Figure 5.Result shows the Sendai virus with natural antigen SeV() compare, Sendai virus antigen peptide composition provided by the invention photoabsorption when detecting Sendai virus positive serum sample is worked as with the full Particle Phase of virus, and it is lower to the detected value of negative serum, illustrate that Sendai virus antigen peptide composition provided by the invention has lower reaction background, specificity is better.Use whole virus particles antigen to do the ELISA criterion that antigen detects, Sendai virus antigen peptide composition provided by the invention can be made and the complete consistent judgement of
particulate antigen17 duplicate samples.Criterion in figure is the OD of all negative control samples
450mean value and 3 times be all negative sample OD
450standard deviation sum,
The detection method accuracy that embodiment 5 Sendai viruses infect detects
Employing comprises the Sendai virus antigen peptide composition of the polypeptide of sequence shown in the polypeptide of sequence shown in the polypeptide, SEQ ID NO:2 with sequence as shown in SEQ ID NO:1 and SEQ ID NO:3, by ELISA method, 10 rats that infect Sendai virus are detected, separately get 5 rats that do not infect Sendai virus as negative control, test the accuracy of Sendai virus method of detecting infection provided by the invention.
First, conventional reagent in preparation ELISA experiment:
Coated damping fluid: get 1.59 grams of Na 2cO 3with 2.93 grams of NaHCO 3be dissolved in distilled water, regulating pH value is 9.6, and adding distil water is to 1000mL;
Lavation buffer solution: get 0.2 gram of KH 2pO 4, 2.9 grams of Na 2hPO 412H 2o, 8.0 grams of NaCl, 0.2 gram of KCl, 0.05% Tween-20 aqueous solution 0.5mL, dissolve and water, and regulating pH value is 7.4, with distilled water, is settled to 1000mL;
Sample diluting liquid: get 0.1 gram of bovine serum albumin (BSA) and be dissolved in the lavation buffer solution that 100mL prepares;
Stop buffer: get distilled water 178.3mL, dropwise add the 21.7ml vitriol oil (volume fraction is 98%);
Substrate buffer solution: get the Na that concentration is 28.4 grams/L 2hPO 425.7mL, concentration are the citric acid 24.3mL of 19.2 grams/L, and distilled water 50mL mixes and regulates pH value to 5.0;
The ELISA experimental procedure that detects Sendai virus antibody is as follows:
The Sendai virus antigen peptide composition comprising that the embodiment of the present invention is provided with the polypeptide of sequence shown in the polypeptide of sequence shown in the polypeptide, SEQ ID NO:2 of sequence as shown in SEQ ID NO:1 and SEQ ID NO:3 is coated with on elisa plate, every hole 100 μ L, the concentration that comprises the Sendai virus antigen peptide composition of the polypeptide of sequence shown in the polypeptide of sequence shown in the polypeptide, SEQ ID NO:2 with sequence as shown in SEQ ID NO:1 and SEQ ID NO:3 is 10 μ g/mL, and 4 â are spent the night.Washings cleaning of enzyme target 3 times, at 37 â with more than sample diluting liquid sealase target 1h.Prepare testing sample, extract the venous blood of rat to be measured, centrifugal collection serum.With sample diluting liquid, press the dilution proportion serum of 1:2, the serum of getting after dilution adds in enzyme plate, every hole 50 μ L, incubated at room 1h.Each sample is coated with 2 holes.Washings cleaning of enzyme target 3 times, adds two of horseradish peroxidase-labeled to resist, and two anti-dilution proportion of pressing 1:10000, add respectively in hand-hole, and every hole 100 μ L, hatch 1h for 37 â, then clean 3 times with washings.Add horseradish peroxidase substrate buffer solution 100 μ L, after colour developing 5min, add stop buffer 100 μ L, color development stopping.By microplate reader, detect OD 450value, adopt Cut off value method judgement detected result;
The standard of Cut off value method judgement is:
Examined the OD of solution
450value
it is positive,
Examined the OD of solution
450value
it is negative,
examined the OD of solution
450value
for suspicious, again survey the OD that is examined solution
450be worth, examined the OD of solution
450value is less than
be judged to feminine gender;
Wherein,
oD for all negative control samples
450mean value, SD is all negative sample OD
450standard deviation; Negative control sample is consistent with the detection method of testing sample.
Adopt method provided by the invention to identify rat infection Sendai virus situation, result is as shown in table 1:
Table 1 the present embodiment is to rat infection Sendai virus qualification result
Experimental result shows, adopt method provided by the invention to identify rat infection Sendai virus situation, accuracy can reach 100%, and detection method provided by the invention is described, be a kind of method that Sendai virus infects of effectively identifying fast, its qualification result accurately and reliably.
The detection method accuracy that embodiment 6 Sendai viruses infect detects
Employing is included in to have as shown in SEQ ID NO:1 and replaces in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or add the Sendai virus antigen peptide composition of the polypeptide that one or more amino-acid residues obtain, by ELISA method, 10 rats that infect Sendai virus are detected, separately get 5 rats that do not infect Sendai virus as negative control, test the accuracy of Sendai virus method of detecting infection provided by the invention.
Wherein, replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:7 having as shown in SEQ ID NO:1 in sequence; Replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:8 having as shown in SEQ ID NO:2 in sequence; Replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:9 having as shown in SEQ ID NO:3 in sequence.
First, conventional reagent in preparation ELISA experiment:
Coated damping fluid: get 1.59g Na 2cO 3with 2.93g NaHCO 3be dissolved in distilled water, regulating pH value is 9.6, and adding distil water is to 1000mL;
Lavation buffer solution: get 0.2g KH 2pO 4, 2.9g Na 2hPO 412H 2o, 8.0g NaCl, 0.2g KCl, 0.05% Tween-20 aqueous solution 0.5mL, dissolve and water, and regulating pH value is 7.4, with distilled water, is settled to 1000mL;
Sample diluting liquid: get 0.1g bovine serum albumin (BSA) and be dissolved in the lavation buffer solution that 100mL prepares;
Stop buffer: get distilled water 178.3mL, dropwise add the 21.7mL vitriol oil (volume fraction is 98%);
Substrate buffer solution: get the Na that concentration is 28.4g/L 2hPO 4the citric acid 24.3mL that 25.7mL, concentration are 19.2g/L, distilled water 50mL mixes and regulates pH value to 5.0;
The ELISA experimental procedure that detects Sendai virus antibody is as follows:
The embodiment of the present invention is provided be included in to have as shown in SEQ ID NO:1 replaces in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or the Sendai virus antigen peptide composition that adds the polypeptide that one or more amino-acid residues obtain are coated with on elisa plate, every hole 100 μ L, be included in to have as shown in SEQ ID NO:1 and replace in sequence, disappearance or add the polypeptide that one or more amino-acid residues obtain, in sequence, replace having as shown in SEQ ID NO:2, disappearance or add polypeptide that one or more amino-acid residues obtain and replace in sequence having as shown in SEQ ID NO:3, disappearance or the concentration of adding the Sendai virus antigen peptide composition of the polypeptide that one or more amino-acid residues obtain are 10 μ g/mL, 4 â are spent the night.Washings cleaning of enzyme target 3 times, at 37 â with more than sample diluting liquid sealase target 1h.Prepare testing sample, extract the venous blood of rat to be measured, centrifugal collection serum.With sample diluting liquid, press the dilution proportion serum of 1:2, the serum of getting after dilution adds in enzyme plate, every hole 50 μ L, incubated at room 1h.Each sample is coated with 2 holes.Washings cleaning of enzyme target 3 times, adds two of horseradish peroxidase-labeled to resist, and two anti-dilution proportion of pressing 1:10000, add respectively in hand-hole, and every hole 100 μ L, hatch 1h for 37 â, then clean 3 times with washings.Add horseradish peroxidase substrate buffer solution 100 μ L, after colour developing 5min, add stop buffer 100 μ L, color development stopping.By microplate reader, detect OD 450value, adopt Cut off value method judgement detected result;
The standard of Cut off value method judgement is:
Examined the OD of solution
450value
it is positive,
Examined the OD of solution
450value
it is negative,
examined the OD of solution
450value
for suspicious, again survey the OD that is examined solution
450be worth, examined the OD of solution
450value is less than
be judged to feminine gender;
Wherein,
oD for all negative control samples
450mean value, SD is all negative sample OD
450standard deviation; Negative control sample is consistent with the detection method of testing sample.
Adopt method provided by the invention to identify rat infection Sendai virus situation, result is as shown in table 2:
Table 2 the present embodiment is to rat infection Sendai virus qualification result
Experimental result shows, adopt method provided by the invention to identify rat infection Sendai virus situation, accuracy can reach 100%, and detection method provided by the invention is described, be a kind of method that Sendai virus infects of effectively identifying fast, its qualification result accurately and reliably.
The preparation method of embodiment 7 Sendai virus vaccines (candy pill)
Get and comprise that the Sendai virus antigen peptide composition of the polypeptide of sequence shown in the polypeptide of sequence shown in the polypeptide, SEQ ID NO:2 with sequence as shown in SEQ ID NO:1 and SEQ ID NO:3 adds pharmaceutically acceptable auxiliary material, adopts ordinary method to make candy pill.
The preparation method of embodiment 8 Sendai virus vaccines (oral solutions)
Get and comprise that the Sendai virus antigen peptide composition of the polypeptide of sequence shown in the polypeptide of sequence shown in the polypeptide, SEQ ID NO:2 with sequence as shown in SEQ ID NO:1 and SEQ ID NO:3 adds pharmaceutically acceptable auxiliary material, adopts ordinary method to make oral solutions.
The preparation method of embodiment 9 Sendai virus vaccines (syrup)
Get and comprise that the Sendai virus antigen peptide composition of the polypeptide of sequence shown in the polypeptide of sequence shown in the polypeptide, SEQ ID NO:2 with sequence as shown in SEQ ID NO:1 and SEQ ID NO:3 adds pharmaceutically acceptable auxiliary material, adopts ordinary method to make syrup.
The preparation method of embodiment 10 Sendai virus vaccines (capsule)
Get to be included in and there is as shown in SEQ ID NO:1 in sequence the polypeptide that replaces, lack or add one or more amino-acid residues and obtain, in sequence, replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is as shown in SEQ ID NO:3 the Sendai virus antigen peptide composition that replaces, lacks or add the polypeptide that one or more amino-acid residues obtain in sequence having as shown in SEQ ID NO:2, add pharmaceutically acceptable auxiliary material, adopt ordinary method to make capsule.
Wherein, replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:7 having as shown in SEQ ID NO:1 in sequence; Replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:8 having as shown in SEQ ID NO:2 in sequence; Replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:9 having as shown in SEQ ID NO:3 in sequence.
The preparation method of embodiment 11 Sendai virus vaccines (powder injection)
Get to be included in and there is as shown in SEQ ID NO:1 in sequence the polypeptide that replaces, lack or add one or more amino-acid residues and obtain, in sequence, replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is as shown in SEQ ID NO:3 the Sendai virus antigen peptide composition that replaces, lacks or add the polypeptide that one or more amino-acid residues obtain in sequence having as shown in SEQ ID NO:2, add pharmaceutically acceptable auxiliary material, adopt ordinary method to make powder injection.
Wherein, replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:7 having as shown in SEQ ID NO:1 in sequence; Replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:8 having as shown in SEQ ID NO:2 in sequence; Replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:9 having as shown in SEQ ID NO:3 in sequence.
The preparation method of embodiment 12 Sendai virus vaccines (injection liquid)
Get to be included in and there is as shown in SEQ ID NO:1 in sequence the polypeptide that replaces, lack or add one or more amino-acid residues and obtain, in sequence, replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is as shown in SEQ ID NO:3 the Sendai virus antigen peptide composition that replaces, lacks or add the polypeptide that one or more amino-acid residues obtain in sequence having as shown in SEQ ID NO:2, add pharmaceutically acceptable auxiliary material, adopt ordinary method to make injection liquid.
Wherein, replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:7 having as shown in SEQ ID NO:1 in sequence; Replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:8 having as shown in SEQ ID NO:2 in sequence; Replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:9 having as shown in SEQ ID NO:3 in sequence.
Embodiment 13 Sendai viruses infect the preparation method of detection kit
Get and comprise that the Sendai virus antigen peptide composition of the polypeptide of sequence shown in the polypeptide of sequence shown in the polypeptide, SEQ ID NO:2 with sequence as shown in SEQ ID NO:1 and SEQ ID NO:3 adopts ordinary method, is coated on enzyme plate.
Obtain solution (10 deal):
Lavation buffer solution: get 0.2gKH 2pO 4, 2.9gNa 2hPO 412H 2o, 8.0gNaCl, 0.2gKCl, 0.05% Tween-20 aqueous solution 0.5mL, dissolve and water, and regulating pH value is 7.4, with distilled water, is settled to 1000mL;
Sample diluting liquid: get 0.1g bovine serum albumin (BSA) and be dissolved in the lavation buffer solution that 100ml prepares;
Stop buffer: get distilled water 178.3mL, dropwise add the 21.7ml vitriol oil (volume fraction is 98%);
Substrate buffer solution: get the Na that concentration is 28.4g/L 2hPO 4the citric acid 24.3ml that 25.7mL, concentration are 19.2g/L, distilled water 50mL mixes and regulates pH value to 5.0;
By the enzyme plate, lavation buffer solution, sample diluting liquid, the stop buffer box substrate buffer solution that are coated with the Sendai virus antigen peptide with the aminoacid sequence as shown in SEQ ID NO:1, divide packing, obtain.
Embodiment 14 Sendai viruses infect the preparation method of detection kit
Get to be included in and there is as shown in SEQ ID NO:1 in sequence the polypeptide that replaces, lack or add one or more amino-acid residues and obtain, adopt ordinary method having as shown in SEQ ID NO:2 to replace, lack or add the polypeptide that one or more amino-acid residues obtain in sequence and there is as shown in SEQ ID NO:3 the Sendai virus antigen peptide composition that replaces, lacks or add the polypeptide that one or more amino-acid residues obtain in sequence, be coated on enzyme plate.
Wherein, replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:7 having as shown in SEQ ID NO:1 in sequence; Replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:8 having as shown in SEQ ID NO:2 in sequence; Replace, lack or add the polypeptide that one or more amino-acid residues obtain and there is the aminoacid sequence as shown in SEQ ID NO:9 having as shown in SEQ ID NO:3 in sequence.
Obtain solution (10 deal):
Lavation buffer solution: get 0.2gKH 2pO 4, 2.9gNa 2hPO 412H 2o, 8.0gNaCl, 0.2gKCl, 0.05% Tween-20 aqueous solution 0.5mL, dissolve and water, and regulating pH value is 7.4, with distilled water, is settled to 1000mL;
Sample diluting liquid: get 0.1g bovine serum albumin (BSA) and be dissolved in the lavation buffer solution that 100ml prepares;
Stop buffer: get distilled water 178.3mL, dropwise add the 21.7ml vitriol oil (volume fraction is 98%);
Substrate buffer solution: get the Na that concentration is 28.4g/L 2hPO 4the citric acid 24.3ml that 25.7mL, concentration are 19.2g/L, distilled water 50mL mixes and regulates pH value to 5.0;
By the enzyme plate, lavation buffer solution, sample diluting liquid, the stop buffer box substrate buffer solution that are coated with the Sendai virus antigen peptide with the aminoacid sequence as shown in SEQ ID NO:1 and SEQ ID NO:2, divide packing, obtain.
Embodiment 15 Sendai viruses infect the use of detection kit
Adopt the Sendai virus infection detection kit that the embodiment of the present invention 13ï½14 any one provide to detect 10 rats that infect Sendai viruses, separately get 5 rats that do not infect Sendai virus as negative control, test the accuracy of Sendai virus method of detecting infection provided by the invention.
Extract the venous blood of rat to be measured, centrifugal collection serum.With the sample diluting liquid in test kit provided by the invention, press the dilution proportion serum of 1:2, the serum of getting after dilution adds in the enzyme plate in test kit provided by the invention, every hole 50 μ L, incubated at room 1h.Each sample is coated with 2 holes.Get washings cleaning of enzyme target in test kit provided by the invention 3 times, add the two anti-of horseradish peroxidase-labeled in test kit provided by the invention, two anti-dilution proportion of pressing 1:10000, add respectively in hand-hole, every hole 100 μ L, hatch 1h for 37 â, then clean 3 times with the washings in test kit provided by the invention.Add the horseradish peroxidase substrate buffer solution 100 μ L in test kit provided by the invention, after colour developing 5min, add the stop buffer 100 μ L in test kit provided by the invention, color development stopping.By microplate reader, detect OD 450value, adopt Cut off value method judgement detected result;
The standard of Cut off value method judgement is:
Examined the OD of solution
450value
it is positive,
Examined the OD of solution
450value
it is negative,
examined the OD of solution
450value
for suspicious, again survey the OD that is examined solution
450be worth, examined the OD of solution
450value is less than
be judged to feminine gender;
Wherein,
oD for all negative control samples
450mean value, SD is all negative sample OD
450standard deviation; Negative control sample is consistent with the detection method of testing sample.
Adopt the Sendai virus infection detection kit that the embodiment of the present invention 13 provides to identify rat infection Sendai virus situation, result is as shown in table 3:
Table 3 adopts Sendai virus provided by the invention to infect detection kit to rat infection Sendai virus qualification result
Experimental result shows, adopt test kit provided by the invention to identify rat infection Sendai virus situation, accuracy can reach 100%, illustrates that Sendai virus provided by the invention infects the Sendai virus infection conditions that detection kit can effectively be identified fast, and its qualification result accurately and reliably.
Sendai virus that other embodiments of the invention provide infects detection kit to the detected result of rat infection Sendai virus similarly, illustrate the invention provides to test kit all can to the infection of animal Sendai virus, identify fast and effectively.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
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