CN118546258B - A fusion peptide of antigenic epitope of avian infectious bronchitis virus and its application in indirect ELISA detection - Google Patents
A fusion peptide of antigenic epitope of avian infectious bronchitis virus and its application in indirect ELISA detection Download PDFInfo
- Publication number
- CN118546258B CN118546258B CN202410710444.8A CN202410710444A CN118546258B CN 118546258 B CN118546258 B CN 118546258B CN 202410710444 A CN202410710444 A CN 202410710444A CN 118546258 B CN118546258 B CN 118546258B
- Authority
- CN
- China
- Prior art keywords
- fusion peptide
- antigen
- indirect elisa
- epitope fusion
- antigenic epitope
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明提供一种鸡传染性支气管炎病毒抗原表位融合肽及其在间接ELISA检测中的应用，所制备的抗原表位融合肽即可以用于制备抗体，也可以作为包被抗原用于制备间接ELISA检测制品。所提供的抗原表位融合肽，其氨基酸序列为SEQ ID NO:2或SEQ ID NO:4。本发明通过生物信息学软件对传染性支气管炎病毒株的S1蛋白进行抗原表位分析后，将多个抗原表位融合得到抗原表位融合肽。特异性试验表明本发明制备的抗原表位融合肽为包被抗原与其他常见病原的阳性血清均不发生交叉反应，仅与对GI‑19型IBV阳性血清产生良好反应，即作为检测抗原具有良好的血清型特异性，而与非GI‑19型IBV抗体交叉反应小。
The present invention provides an antigenic epitope fusion peptide of infectious bronchitis virus of chickens and its application in indirect ELISA detection. The prepared antigenic epitope fusion peptide can be used to prepare antibodies, and can also be used as a coating antigen to prepare indirect ELISA detection products. The provided antigenic epitope fusion peptide has an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. After the present invention performs antigenic epitope analysis on the S1 protein of the infectious bronchitis virus strain by bioinformatics software, multiple antigenic epitopes are fused to obtain the antigenic epitope fusion peptide. Specificity tests show that the antigenic epitope fusion peptide prepared by the present invention is a coating antigen and does not cross-react with positive sera of other common pathogens, but only reacts well with positive sera of GI-19 type IBV, that is, it has good serotype specificity as a detection antigen, and has little cross-reaction with non-GI-19 type IBV antibodies.
Description
Technical Field
The invention belongs to the technical field of vaccine antigen preparation, and particularly relates to a chicken infectious bronchitis virus epitope fusion peptide and application thereof in indirect ELISA detection.
Background
Infectious bronchitis (Infectious bronchitis, IB) is an acute, highly contagious, viral respiratory disease caused by infectious bronchitis virus (Infectious bronchitis virus, IBV). Chickens of various ages and different varieties are easy to infect the infectious bronchitis of the chickens, particularly chickens of 1-4 weeks old are most serious, and the death rate is 40% -90%. Although vaccine prophylaxis is widely used, IB is not effectively controlled and variant or new genotypes continue to appear. IB has occurred and is prevalent in most parts of the world today, but there is a difference in the genotypes or epidemic strains that are prevalent in different areas. Up to now, there are a minimum of 3 genotypes in our country, GI-19, GVI and GVII respectively. Among them, GI-19 type, namely QX type, is found in China at the earliest, is the most dominant IBV genotype currently popular in China, and causes great loss for poultry industry in China.
IBV is a single-stranded positive strand RNA virus of about 27.6kb in genome, belonging to the genus gamma coronavirus. The virus particles are similar to spheres and have the diameter of 82nm, and the virus particles consist of 4 structural proteins of fiber (S) protein, membrane (M) glycoprotein, nucleocapsid (N) protein and small membrane (E) protein.
The laboratory methods currently used for IB antibody detection mainly include methods such as enzyme-linked immunosorbent assay (ELISA), agar diffusion precipitation Assay (AGP) and virus neutralization assay (VN). The 3 detection methods have better specificity, but the AGP sensitivity is poorer, the neutralization test operation is complex and time-consuming, and requires higher laboratory conditions, and the ELISA method is suitable for laboratory serum diagnosis and large-scale epidemic investigation due to the fast speed, low requirements on the experimental conditions, no need of aseptic operation and capability of detecting a large number of samples in a short time.
Disclosure of Invention
The invention provides an antigen epitope fusion peptide of chicken infectious bronchitis virus and application thereof in indirect ELISA detection, and the prepared antigen epitope fusion peptide can be used for preparing antibodies or used as a coating antigen for preparing indirect ELISA detection products.
The antigen epitope fusion peptide of the infectious bronchitis virus provided by the invention is antigen epitope fusion peptide S1-I, the amino acid sequence of which is SEQ ID NO. 2, and the corresponding sequence of one coding nucleotide is SEQ ID NO. 1;
The antigen epitope fusion peptide of the infectious bronchitis virus provided by the invention is antigen epitope fusion peptide S1-II, the amino acid sequence of which is SEQ ID NO. 4, and the corresponding sequence of one coding nucleotide is SEQ ID NO. 3;
Referring to the escherichia coli codon preference table, codon optimization is carried out, the nucleotide sequence of the optimized S1-I is SEQ ID NO. 5, the nucleotide sequence of the S1-II is SEQ ID NO. 6,
The invention also provides a recombinant expression vector, wherein a nucleic acid fragment for encoding the antigen epitope fusion peptide is inserted into the recombinant expression vector;
the invention also provides a prokaryotic expression host bacterium, wherein the prokaryotic expression host bacterium carries the recombinant expression vector;
the invention also provides application of the chicken infectious bronchitis virus epitope fusion peptide as a coating antigen;
As a specific record of the examples, the application is for the preparation of indirect ELISA detection preparations;
the invention also provides an indirect ELISA detection product, wherein the coating antigen is the chicken infectious bronchitis virus epitope fusion peptide;
further, the antigen coating concentration is 0.5-1 mug/mL.
In a further aspect, the invention provides an indirect ELISA method for detecting chicken infectious bronchitis virus, which is to use the indirect ELISA detection product to detect, wherein the S1-I antigen coating concentration is 1 mug/mL, the serum dilution is 1:1000, the secondary antibody dilution is 1:10000, and 5% skim milk is the optimal blocking solution.
Recombinant protein S1-II is used as a coating antigen, the antigen coating concentration is 0.5 mug/mL, the serum dilution is 1:1000, the secondary antibody dilution is 1:5000, and 5% skim milk is the optimal sealing liquid.
The invention carries out antigen epitope analysis on the S1 protein of the infectious bronchitis virus strain through bioinformatics software, and then fuses a plurality of antigen epitopes to obtain antigen epitope fusion peptides. The specificity test shows that the antigen epitope fusion peptide prepared by the invention is a coating antigen and does not have cross reaction with positive serum of other common pathogens, and only has good reaction with GI-19 type IBV positive serum, namely has good serotype specificity as a detection antigen, but has small cross reaction with non-GI-19 type IBV antibodies.
Drawings
FIG. 1 is a schematic diagram of the sequence structures of epitope polypeptides S1-I and S1-II,
FIG. 2 is a graph showing the results of S1-I sequence analysis,
FIG. 3 is a graph showing the results of S1-II sequence analysis;
FIG. 4 shows a prokaryotic expression vector double-restriction enzyme map, wherein M is a relative molecular mass standard (5000 bp) of nucleic acid, lanes 1-2 are pET-30a (+) -S1-I double-restriction enzyme products, and lanes 3-4 are pET-30a (+) -S1-II double-restriction enzyme products;
FIG. 5 shows a recombinant protein S1-I/II expression purification electrophoresis detection chart, wherein FIG. A shows a recombinant protein S1-I expression purification electrophoresis detection chart, FIG. B shows a recombinant protein S1-II expression purification electrophoresis detection chart, M shows a protein relative molecular mass standard (180 kDa), lane 1 shows a concentrated recombinant protein S1-I, and lane 2 shows a concentrated recombinant protein S1-II;
FIG. 6 shows a positive serum binding strain situation diagram after recombinant protein immunization, wherein the diagram A shows an S1-I positive serum binding strain situation diagram, the diagram B shows an S1-II positive serum binding strain situation diagram, M shows a protein relative molecular mass standard (180 kDa), lane 1 shows a GI-19 type IBV strain sample, and lane 2 shows a GI-19 type IBV strain sample;
FIG. 7 is a graph showing the neutralization level identification of the production of the recombinant protein S1-I/II by the organism;
FIG. 8 is a graph showing the judgment of the indirect ELISA positive values, wherein the graph A shows the judgment of the indirect ELISA positive values of the recombinant protein S1-I and the graph B shows the judgment of the indirect ELISA positive values of the recombinant protein S1-II;
FIG. 9 is a graph showing indirect ELISA sensitivity assay, wherein FIG. A shows an indirect ELISA sensitivity assay for recombinant protein S1-I and FIG. B shows an indirect ELISA sensitivity assay for recombinant protein S1-II.
Detailed Description
The invention uses various bioinformatics to analyze the antigenicity of the IBV S1 protein, designs and selects dominant antigen epitope on the S1 gene, synthesizes after codon optimization, constructs prokaryotic expression plasmid, converts plasmid into competent cells such as E.coli Rosetta, E.coli BL21 (DE 3) and the like to carry out prokaryotic expression and purify recombinant protein, further uses the purified protein as antigen coated ELISA plate, and respectively detects IBV (GVI-1), IBV (H120), IBV (ahysx-1), AIV (H5), AIV (H9), NDV and GAstV positive serum, preferably selects S1-I recombinant protein with the best effect of reflecting QX positive serum and weaker effect of reflecting other non-GI-19 positive serum as detection antigen, and establishes ELISA method for detecting the immune antibody of GI-19 type IB vaccine and clinically monitoring IBV infection.
The information of plasmids, strains and serum in the embodiment of the invention is as follows:
S1-I and S1-II sequences are subjected to codon optimization and then sent to Qingdao Ruibo Ke Xingxing biological Co., ltd for synthesis, E.coli Rosetta, E.coli BL21 (DE 3) competent cells are purchased from Beijing Bomaide gene technology Co., ltd, and IBV (GI-19), IBV (GVI-1), IBV (H120), IBV (ahysx-1) AIV (H5), AIV (H9), NDV and GAstV positive serum and negative serum are all stored in a laboratory.
IPTG solution, ELISA coating solution, stop solution, two-component TMB developing solution are purchased from Beijing Soilebao technologies, inc., horseradish perhydride enzyme (HRP) marked goat anti-chicken IgG is purchased from Beijing Boaosen biotechnology, inc., plasmid miniprep kit is purchased from Beijing Tiangen Biochemical technologies, inc., 96-hole removable ELISA plate is purchased from Wuxi Jiujingshi life technologies, inc., and primer synthesis and sequencing are completed by Qingdao Rui Boke Xingxing biological, inc.
The present invention will be described in detail with reference to the following examples and the accompanying drawings.
Example 1 screening and sequence optimization of epitope Polypeptides
The S1 gene fragment of IBV GI-19 strain (GenBank accession number MN 548289) was analyzed for epitopes using an online tool to log in the website http:// imaged. Med. Ucm. Es/Tools/anti-pl prediction (Table 1). The two sections of antigen polypeptide epitopes S1-I and S1-II (figure 1) with strong antigenicity, good hydrophilicity and good flexibility are constructed by spacing the antigen epitope sequences by an alanine-tyrosine (AAY) connecting sequence, and the secondary structure, antigenicity index, hydrophilicity and flexibility of the multi-epitope peptide are analyzed by applying DNAStar protein software. The secondary structure, antigenicity index, hydrophilicity and flexibility of S1-I and S1-III are shown in figures 2 and 3, and the antigenicity of the two polypeptides is good.
TABLE 1 prediction of IBV S1 epitope
Wherein the corresponding nucleotide sequence of S1-I is as follows (SEQ ID NO: 1):
AAGTCACTGTTTTTAGTGACCATTTTGTGTGCACTATGTAGTGCAAATGCTGCTTACAATAATTATGTGTACTACTACCAAAGTGCTGCTGCTTACCTACAAGGAGGTGCTTATGCAGTAGTCAATGCTGCTTACTCTGCACATCAGTGCACTGTTGGTGTTATTAAGGATGTTTATAATCAAAGTGTGGCTTCCATAGCTATGACAGCACCTCTTGCTGCTTACTCTAAGTCACAATTCTGTAGTGCACACTGTAATTTTTCTGAAATTACAGTTTTTGTCACACATTGTTATAGTGCTGCTTACGGTAGTGGGTCTTGTCCTATAACAGCTGCTTACTTATTTTATAATTTAACAGTTAGCGTATCTAAAGCTGCTTACAAATCTTTTCAATGTGTTAACAACTTCACATCTGTTTATTTAAATGGTGATCTTGTTTTTACTGCTGCTTACGATGTTACGTCAGCAGGTGTGTATTTTAAAGCAGCTGCTTACAAAGAATTTAAGGTTCTTGCTTACTTTGTTAATGCTGCTTACGCACAAGATGTAGTTTTGTGCGACGCTGCTTACCCCAAGGGTTTGCTAGCTTGTCAAGCTGCTTACACTAATAGTACTTTGGTTAGGGAAAAGTTCATCGTCTATCGCGAAGCTGCTTACGTTAATACTACTCTGGCGTTAACTGCTGCTTACGTTAATACTTTTCATTTATACCAAACAGCTGCTTACTATAATTTTAATTTGTCATTTCTGAGTCAGTTTGTGTATAAG;
the corresponding amino acid sequence of S1-I is as follows (SEQ ID NO: 2):
KSLFLVTILCALCSANAAYNNYVYYYQSAAAYLQGGAYAVVNAAYSAHQCTVGVIKDVYNQSVASIAMTAPLAAYSKSQFCSAHCNFSEITVFVTHCYSAAYGSGSCPITAAYLFYNLTVSVSKAAYKSFQCVNNFTSVYLNGDLVFTAAYDVTSAGVYFKAAAYKEFKVLAYFVNAAYAQDVVLCDAAYPKGLLACQAAYTNSTLVREKFIVYREAAYVNTTLALTAAYVNTFHLYQTAAYYNFNLSFLSQFVYK;
The corresponding nucleotide sequence of S1-II is as follows (SEQ ID NO: 3):
TCTGCACATCAGTGCACTGTTGGTGTTATTAAGGATGTTTATAATCAAAGTGTGGCTTCCATAGCTATGACAGCACCTCTTGCTGCTTACTCTAAGTCACAATTCTGTAGTGCACACTGTAATTTTTCTGAAATTACAGTTTTTGTCACACATTGTTATAGTGCTGCTTACGGTAGTGGGTCTTGTCCTATAACAGCTGCTTACTTATTTTATAATTTAACAGTTAGCGTATCTAAAGCTGCTTACAAATCTTTTCAATGTGTTAACAACTTCACATCTGTTTATTTAAATGGTGATCTTGTTTTTACTGCTGCTTACGATGTTACGTCAGCAGGTGTGTATTTTAAAGCAGCTGCTTACAAAGAATTTAAGGTTCTTGCTTACTTTGTTAATGCTGCTTACGCACAAGATGTAGTTTTGTGCGACGCTGCTTACCCCAAGGGTTTGCTAGCTTGTCAAGCTGCTTACACTAATAGTACTTTGGTTAGGGAAAAGTTCATCGTCTATCGCGAAGCTGCTTACGTTAATACTACTCTGGCGTTAACTGCTGCTTACGCTGCTTACGTTAATACTTTTCATTTATACCAAACAGCTGCTTACTATAATTTTAATTTGTCATTTCTGAGTCAGTTTGTGTATAAGGCTGCTTACTATGGGTCCTACCACCCTAGTTGTTCTTTTGCTGCTTACTGGTTTAATTCCTTGTCAGTTTCTCTTACTTATGGACCCCTACAGGGAGGGTGTAAGCAATCTGTTTTTAGTGGTAAGGCAACGTGTTGTTACGCCTACTCTTATGCTGCTTACGGCCCAATGGCATGTAAAGGTGTTTATTCAGGTGCTGCTTACTTTGAATGTGGATTGCTGGTTTATGTTACTGCTGCTTACACAGAGCCCTTAGTATTAACGCAAGCTGCTTACGGTGCCATAGATGTTTTTGTTGTACAGGGCATCTATGGTCTTAATTATTACAAGGTTAATCCTTGTGAAGAT.
the corresponding amino acid sequence of S1-II is as follows (SEQ ID NO: 4):
SAHQCTVGVIKDVYNQSVASIAMTAPLAAYSKSQFCSAHCNFSEITVFVTHCYSAAYGSGSCPITAAYLFYNLTVSVSKAAYKSFQCVNNFTSVYLNGDLVFTAAYDVTSAGVYFKAAAYKEFKVLAYFVNAAYAQDVVLCDAAYPKGLLACQAAYTNSTLVREKFIVYREAAYVNTTLALTAAYAAYVNTFHLYQTAAYYNFNLSFLSQFVYKAAYYGSYHPSCSFAAYWFNSLSVSLTYGPLQGGCKQSVFSGKATCCYAYSYAAYGPMACKGVYSGAAYFECGLLVYVTAAYTEPLVLTQAAYGAIDVFVVQGIYGLNYYKVNPCED.
with reference to the E.coli codon preference table, codon optimization was performed, and the nucleotide sequence of the optimized S1-I was as follows (SEQ ID NO: 5):
AAAAGCCTGTTCCTGGTTACCATCCTGTGCGCCCTGTGTTCTGCCAACGCGGCATACAATAACTACGTGTACTACTACCAGAGCGCGGCTGCTTACCTGCAAGGTGGCGCTTACGCTGTCGTAAACGCTGCATACTCCGCTCATCAGTGCACCGTTGGTGTAATTAAGGATGTTTACAACCAGTCCGTAGCATCCATTGCGATGACCGCGCCGCTGGCTGCATATTCTAAGTCCCAGTTCTGCTCCGCGCACTGCAATTTCAGCGAGATTACTGTTTTCGTAACCCACTG CTACTCCGCAGCGTATGGCTCTGGTAGCTGCCCGATCACTGCTGCCTACCTGTTCTATAACCTGACCGTGAGCGTGTCTAAAGCCGCGTATAAATCTTTCCAGTGCGTGAACAACTTCACCTCCGTTTACCTGAACGGTGACCTGGTTTTTACTGCCGCCTACGATGTAACCTCTGCCGGTGTTTATTTCAAGGCAGCGGCGTACAAAGAATTCAAGGTGCTGGCGTACTTCGTCAACGCCGCTTATGCCCAGGATGTTGTGCTGTGTGACGCAGCGTACCCGAAAGGTCTGCTGGCGTGTCAGGCAGCGTACACGAATTCTACCCTGGTACGCGAGAAATTCATTGTCTACCGTGAGGCTGCTTACGTCAACACCACTCTGGCACTGACCGCAGCCTACGTGAACACCTTCCATCTGTATCAGACTGCGGCGTACTACAACTTCAACCTGTCCTTTCTGAGCCAGTTCGTGTACAAA;
the nucleotide sequence of the optimized S1-II is as follows (SEQ ID NO: 6):
TCTGCCCACCAGTGCACCGTTGGCGTGATTAAAGACGTTTACAACCAGTCCGTTGCTAGCATTGCAATGACTGCCCCGCTGGCAGCCTACTCCAAAAGCCAGTTTTGTTCCGCGCACTGCAACTTCTCTGAGATCACCGTATTTGTTACCCATTGTTATTCTGCGGCCTATGGCTCTGGTAGCTGTCCGATCACGGCGGCGTATCTGTTTTATAACCTGACCGTCAGCGTTTCCAAAGCGGCTTACAAGTCCTTCCAGTGCGTCAACAATTTCACCTCTGTTTATCTGAACGGTGACCTGGTGTTTACTGCGGCATATGACGTAACCAGCGCTGGCGTTTATTTTAAAGCGGCGGCGTACAAAGAATTCAAAGTACTGGCATACTTCGTCAACGCAGCGTACGCTCAGGATGTAGTTCTGTGCGACGCAGCATACCCGAAAGGTCTGCTGGCTTGTCAGGCTGCGTACACTAACTCCACCCTGGTTCGCGAAAAGTTCATCGTATACCGCGAAGCGGCTTACGTAAACACGACTCTGGCACTGACCGCAGCTTACGCTGCTTACGTAAACACGTTCCACCTGTACCAAACCGCGGCCTACTACAACTTCAATCTGAGCTTTCTGAGCCAGTTCGTATACAAGGCTGCCTACTACGGCTCCTACCACCCGTCTTGCTCTTTCGCTGCATACTGGTTCAACAGCCTGTCTGTCTCCCTGACCTACGGCCCGCTGCAGGGTGGCTGCAAACAGAGCGTCTTCTCCGGTAAAGCGACCTGCTGTTATGCATACTCCTATGCAGCGTATGGTCCGATGGCGTGTAAAGGTGTTTACTCCGGCGCCGCCTACTTTGAGTGCGGTCTGCTGGTATATGTCACCGCGGCTTATACTGAACCTCTGGTCCTGACCCAGGCGGCATACGGCGCTATTGACGTATTCGTTGTTCAGGGCATCTATGGCCTGAACTATTACAAGGTGAACCCGTGTGAAGAT.
EXAMPLE 2 construction of prokaryotic expression vectors and recombinant expression of epitope Polypeptides
1. Construction of recombinant expression plasmid vectors
2 Pairs of primers are designed and synthesized according to the optimized sequence, and specific sequence information is as follows:
S1-I-F:5′-AAGAATTCAAAAGCCTGTTCCTGGTTAC-3′,
S1-I-R:5′-TTCTCGAGTTTGTACACGAACTGGCTCA-3′;
S1-II-F:5′-AAGAATTCTCTGCCCACCAGTGCACCGT-3′,
S1-II-R5'-TTCTCGAGATCTTCACACGGGTTCACCT-3' (underlined indicates EcoRI and XhoI cleavage sites introduced respectively), its theoretical length S1-I is 768bp, and S1-II is 990bp. Using plasmid containing S1-I and S1-II genome as template Max DNA Polymerase kit for PCR amplification. The reaction system was 2 XBuffer 10. Mu.L, 1. Mu.L each for the upstream and downstream primers, 1. Mu.L for the plasmid, and RNASE FREE H 2 O7. Mu.L. The reaction conditions were 98℃for 2min,98℃for 10s,52℃for 15s,72℃for 1min,35 cycles, 72℃for 10min and 4℃for termination. And (3) after agarose gel electrophoresis of PCR amplified products, recovering and purifying DNA by using a gel recovery kit, simultaneously carrying out double digestion and recovery purification on target gene fragments and pET30a (+) vectors by using EcoRI and XhoI restriction enzymes, connecting T4DNA ligase at 16 ℃ overnight, converting the connected products into DH5 alpha competent cells, extracting plasmid DNA by using a plasmid extraction kit, and carrying out sequencing identification by using Qingdao Rui Boke biological limited company after the double digestion identification of EcoRI and XhoI is correct.
The recombinant prokaryotic expression plasmid pET-30a (+) -S1-I, pET-30a (+) -S1-II is subjected to double digestion by using designed restriction enzymes EcoRI and XhoI to identify a target gene, and after double digestion, the length of S1-I of 7688 bp, S1-II of 990bp and 5400bp is obtained, and the fragment and the vector are respectively consistent with the target fragment and the insert vector (figure 4).
2. Inducible expression of genes and purification and concentration of expression products
Plasmid was extracted from pET-30a (+) -S1-I, pET-30a (+) -S1-II bacteria solution, transferred into E.coli Rosetta and E.coli BL21 (DE 3) competent cells, 50. Mu.L was spread on LB agar plates containing kan (100. Mu.g/mL), cultured overnight at 37℃and single white colonies were picked up and shake-cultured overnight in LB liquid medium containing kan. Then amplifying shake culture according to the ratio of 1:100, adding IPTG to the final concentration of 0.8mmol/L when the OD value reaches about 0.4-0.6, and shake culturing at 28 ℃ for 12h. The bacterial cells were disrupted by sonication, the disrupted bacterial solution was centrifuged at 4℃and 10000rmp/min for 10min, and the supernatant and precipitate of the lysate were separated and analyzed by SDS-PAGE for the expression form of the recombinant protein. Removing the impurity protein under the denaturation condition by adopting a method of washing inclusion bodies, and dialyzing the denatured protein under different urea concentrations for gradual renaturation. Purified proteins were detected by SDS-PAGE and Western blot, and the concentration of the purified proteins was determined using a nucleic acid protein meter. The protein of the large induced expression was purified using His Trap TM affinity chromatography column. Purification effect the purified proteins obtained by SDS-PAGE analysis were designated S1-I, S1-II, respectively. Placing the purified protein in a dialysis bag, placing the dialysis bag filled with recombinant protein in a protein dialysate for renaturation, sequentially carrying out gradient dialysis on the protein dialysate containing urea with different concentrations (8 mmol/L, 6mmol/L, 4mmol/L, 2mmol/L and 1 mmol/L) from high to low at intervals of 12 hours, replacing the protein dialysate with the next gradient protein dialysate, and finally placing the protein dialysate in PBS for dialysis for 12 hours. Protein concentration is carried out by utilizing a sucrose embedding dialysis bag, and renaturated purified protein is identified by utilizing Western-blot and SDS-PAGE.
A large amount of the recombinant protein S1-I, S-II which is induced to be expressed is concentrated by urea gradient dialysis. The renaturated purified protein is identified by adopting Western blot and SDS-PAGE methods. The results showed that purer recombinant proteins were obtained and that both recombinant proteins reacted with GI-19 type IBV positive serum to generate specific bands (fig. 5).
Example 3 recombinant proteins S1-I, S-II as coating antigen
1. Preparation of GI-19 positive serum
SPF chicken was immunized for the first time at 14 days of age, with white oil as adjuvant, and leg intramuscular injection of 100ng recombinant protein S1-I/II, with two weeks apart of booster immunization at the same immunization dose, and serum was collected weekly. The GI-19 type IBV strain stored in a laboratory is diluted to 10 3EID50/0.1 ml for preparing a Western-blot sample, and the generation of antibodies in the organism is primarily judged through the Western-blot. Western-blot results show that antibodies can bind to the strain samples. Virus neutralization experiments were performed to determine the neutralizing antibody level of serum, the strain was diluted to 200EID 50/0.1 mL with PBS, mixed with collected serum, neutralized at room temperature for 1h, 10-11 day old SPF chick embryos inoculated in the allantoic cavity, 0.2mL per embryo, and placed at 37 ℃ for 144h. The chicken blood with the neutralizing antibody level reaching 6Log 2 is collected by a heart blood sampling mode, incubated for 1h at 37 ℃, centrifuged for 10min at 4000rmp/min after incubation for 1h at 4 ℃, and the collected serum is packaged and stored at-20 ℃ for standby.
The GI-19 type IBV strain stored in a laboratory is diluted to 10 3EID50/0.1 ml for preparing a Western-blot sample, positive serum is diluted to 1:200, and detection is carried out by a Western-blot method, and a Western-blot result shows that recombinant protein S1-I/II is injected into SPF chickens, so that antibodies can be stimulated to be produced by organisms (figure 6). The detection result of the neutralizing antibody shows that the highest neutralizing antibody of S1-I serum can reach 6Log 2 (3/5), and the residual SPF chicken antibody level is 5Log 2(1/5)、4Log2 (1/5). The neutralizing antibodies of S1-II serum were up to 6Log 2 (2/5), and the residual SPF chicken neutralizing antibody level was 5Log 2((2/5)、4Log2 (1/5) (FIG. 7).
2. Establishment of an Indirect ELISA detection method
1) Optimization of antigen coating concentration, antiserum and enzyme-labeled antibody dilution
The chessboard titration method determines the coating concentration of the recombinant expressed S1-I, S-II epitope polypeptide and the dilution of an antiserum. Purified S1-I, S1-II epitope polypeptide was coated on the ELISA plate at 4℃for 1h at a total of 4 concentrations of 2. Mu.g/L, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL. After three 1 XPBST washes, 5% skim milk was blocked at 37℃for 1h. After three PBST washes, positive and negative sera were added to the elisa plate at 8 dilutions of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and reacted for 1h at 37 ℃. The plates were washed with 1 XPBST and incubated at 37℃for 1h with HRP-labeled rabbit anti-chicken IgY antibody (1:5000 dilution, 1:10000 dilution). After washing three times with 1 XPBST, color development was performed with 100. Mu.L TMB solution. The reaction was stopped with 50. Mu.L ELISA stop solution. Absorbance was measured at OD450 using a microplate reader. The optimal serum dilution and antigen coating concentration were determined from the P/N maximum.
The results of chessboard titration tests on positive and negative sera using recombinant protein S1-I as coating antigen showed (Table 2) that P/N was 28.178 maximum when the antigen coating concentration was 1. Mu.g/mL, the serum dilution was 1:1000, and the secondary antibody dilution was 1:10000. The conditions were thus chosen as the reaction conditions for the subsequent experiments.
TABLE 2 determination of recombinant protein S1-I coating concentration, serum dilution and optimal second antibody dilution
The results of chessboard titration tests on positive and negative sera using recombinant protein S1-II as coating antigen showed (Table 3) that P/N was 14.917 max when the antigen coating concentration was 0.5 μg/mL, the serum dilution was 1:1000, and the secondary antibody dilution was 1:5000. The conditions were thus chosen as the reaction conditions for the subsequent experiments.
TABLE 3 determination of recombinant protein S1-II coating concentration, serum dilution and optimal second antibody dilution
2) Determination of ELISA blocking solution
The coated ELISA plates of S1-I, S1-II were blocked with 1% BSA, 2% BSA, 5% skim milk and 10% skim milk, respectively, at 37℃for 1h. After three washes of 1 XPBST, the best dilutions of positive and negative sera were used as primary antibodies and reacted for 1h at 37 ℃. After three washes with 1 XPBST, HRP-labeled rabbit anti-chicken IgY antibodies were incubated at 37℃for 1h at 1:10000. After washing three times with 1 XPBST, 100. Mu.L of TMB substrate chromogenic solution was added and the reaction was stopped with 50. Mu.L of ELISA stop solution. Absorbance was measured at OD450 using a universal microplate reader. Based on the P/N maximum, the optimal ELISA blocking solution and enzyme-labeled antibody dilutions were determined.
ELISA test results (Table 4) using recombinant protein S1-I as coating antigen and 1% BSA, 2% BSA, 5% skim milk and 10% skim milk as blocking solution revealed that the P/N value was 33.844 maximum when the ELISA plate was blocked with 10% skim milk. Thus, 5% skim milk was determined to be the optimal confining liquid.
TABLE 4 determination of the blocking conditions for recombinant protein S1-I as coating antigen
ELISA test results (Table 5) using recombinant protein S1-II as coating antigen and 1% BSA, 2% BSA, 5% skim milk and 10% skim milk as blocking solution revealed that the P/N value was 13.015 maximum when the ELISA plate was blocked with 10% skim milk. Thus, 5% skim milk was determined to be the optimal confining liquid.
TABLE 5 determination of the blocking conditions for recombinant protein S1-II as coating antigen
3) Determination of reaction time of primary and secondary antibodies
The coated S1-I/II ELISA plates were blocked with 10% skim milk at 37℃for 1 h. After three washes of 1 XPBST, the best dilutions of positive and negative serum were used as primary antibodies and incubated at 37℃for 0.5,1h,1.5h and 2h, respectively. After three washes of 1 XPBST, HRP-labeled rabbit anti-chicken IgY antibodies were diluted in a ratio of 1:10000 and incubated at 37℃for 0.5h,1h,1.5h and 2h, respectively. After washing three times with 1 XPBST, 100. Mu.L of TMB substrate chromogenic solution was added and the reaction was stopped with 50. Mu.L of ELISA stop solution. Absorbance was measured at OD450 using a microplate reader. And determining the reaction time of the primary antibody and the enzyme-labeled secondary antibody according to the maximum value of P/N.
Determination of antiserum and enzyme-labeled antibody reaction time
Recombinant protein S1-I was used as coating antigen, and the P/N value was 31.857 max when a primary antiserum was incubated for 1h and the reaction time of the enzyme-labeled antibody was 1h (Table 6). Therefore, the optimal incubation time of the primary antibody and the reaction time of the enzyme-labeled antibody are determined to be 1h.
TABLE 6 determination of the blocking conditions for recombinant protein S1-I as coating antigen
Recombinant protein S1-II was used as coating antigen, and the P/N value was 16.948 max when a primary antiserum was incubated for 0.5h and the reaction time of the enzyme-labeled antibody was 1h (Table 7). Therefore, the optimal incubation time of the primary antibody and the reaction time of the enzyme-labeled antibody are determined to be 1h.
TABLE 7 determination of the blocking conditions for recombinant protein S1-II as coating antigen
4) Indirect ELISA negative and positive judgment standard
24 Chicken sera were selected, positive controls were set, OD 450nm values of negative and positive sera were determined according to the optimized indirect ELISA assay, and the mean (X) and Standard Deviation (SD) of the N/P values were calculated. When S/P is more than or equal to X+3SD, the test result is positive, and when S/P is less than X+3SD, the test result is negative.
The recombinant protein S1-I is taken as a coating antigen, the OD 450 nm value of 24 parts of serum at the dilution of 1:2000 is counted, the X=0.064 and S=0.011 of all negative serum OD 450 nm are calculated, the critical value of S/P=0.097 is calculated as the critical value of negative judgment, the positive judgment is carried out when the S/P is more than or equal to 0.097, and the negative judgment is carried out when the S/P is less than 0.097 (figure 8A). The recombinant protein S1-II is taken as a coating antigen, the OD450 nm value of 24 serum at the dilution of 1:2000 is counted, X=0.070 and S=0.022 of all negative serum OD450 nm are calculated, the critical value of S/P=0.136 is calculated as the critical value of negative judgment, the positive judgment is carried out when the S/P is more than or equal to 0.136, and the negative judgment is carried out when the S/P is less than 0.136 (figure 8B). Experimental results show that the detection lower limit of the recombinant protein S1-I serving as the coating antigen is lower.
5) Repeatability test
The intra-batch reproducibility test was performed using 3 batches of the antigen-coated ELISA plates of the same batch, and the inter-batch reproducibility test was performed using 3 batches of the antigen-coated ELISA plates of different batches. Each time an indirect ELISA assay was performed with 6 sera, 3 replicates were set per serum, OD 450nm values were read, X, S and Coefficient of Variation (CV) were calculated, and the results were counted and analyzed.
The recombinant protein S1-I was used as a coating antigen for both intra-and inter-batch reproducibility tests, and showed that 5 parts of the positive serum from the reproducibility test had an intra-batch Coefficient of Variation (CV) of 1.08% at a minimum, 5.55% at a maximum, and an inter-batch CV of 1.64% at a minimum, 5.64% at a maximum, each less than 10% (Table 8). The results of the intra-and inter-batch reproducibility tests with recombinant protein S1-II as coating antigen showed that 5 parts of the positive serum from the reproducibility test had an intra-batch CV of 2.12% at a maximum of 5.27% and an inter-batch CV of 1.56% at a minimum of 5.38% and less than 10% each (Table 9). The results show that the experiment using the recombinant protein S1-I as the coating antigen has better repeatability.
TABLE 8 results of within-and inter-batch repeatability of recombinant proteins S1-I
TABLE 9 results of within-and inter-batch repeatability of recombinant protein S1-II
6) Sensitivity test
The antibody titer of the positive serum is detected by diluting the GI-19 type IBV antibody positive serum from 1:1000 to 1:128000 times to determine the positive judgment standard.
Recombinant protein S1-I was used as a coating antigen for sensitivity experiments, IBV positive serum was diluted from 1:1000 fold for indirect ELISA detection, and OD450 value was still higher than the decision criteria when primary antiserum was diluted 1:32000 (FIG. 9A). Recombinant protein S1-II was used as coating antigen for sensitivity experiments, IBV positive serum was diluted from 1:1000 fold for indirect ELISA detection, and OD450 value was still higher than the decision criteria when primary antiserum was diluted 1:16000 (FIG. 9B). The recombinant protein S1-I is used as a coating antigen, so that the sensitivity is higher.
7) Specificity test
The established indirect ELISA method is used for respectively detecting positive serum of AIV (H5), AIV (H9), NDV, GAstV, IBV (GVI-1), IBV (H120) and IBV (ahysx-1), and simultaneously setting positive serum and negative serum of IBV for contrast, and repeating each sample for 3 times to judge whether the method has cross reaction or not.
The results showed that the recombinant protein S1-I was used as a coating antigen for indirect ELISA detection of AIV (H5), AIV (H9), NDV, GAstV positive serum and SPF serum, and that the S/P values of the other serum except the IBV positive serum were all lower than the threshold value of 0.097 (Table 10). The recombinant protein S1-I is coating antigen, and the indirect ELISA detection is carried out on AIV (H5), AIV (H9), NDV, GAstV positive serum and SPF serum, and the S/P value of other serum except the IBV positive serum is lower than the critical value of 0.136 (Table 11). The recombinant protein S1-I is taken as a coating antigen and only well reacts with the positive serum of the type GI-19 IBV, the P/N value of the positive serum detection result of the type GVI-1 IBV, the type H120 IBV and the type ahysx-1 IBV is lower, and the result shows that the S1-I has good serotype specificity as a detection antigen and has small cross reaction with the non-GI-19 IBV antibody.
TABLE 10 recombinant protein S1-I specificity test results Table
TABLE 11 recombinant protein S1-II specificity test results Table
In conclusion, the genetic engineering recombinant GI-19 type IBV epitope polypeptide S1-I provided by the invention can be specifically combined with GI-19 type IBV positive serum, has weak cross reaction with non-GI-19 type IBV positive serum, and can be used for detection and evaluation of immune antibodies of GI-19 type IB vaccine and clinical GI-19 type IBV infection monitoring.
Claims (8)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411573590.7A CN119320459B (en) | 2024-06-03 | 2024-06-03 | Avian infectious bronchitis virus epitope fusion peptide |
| CN202410710444.8A CN118546258B (en) | 2024-06-03 | 2024-06-03 | A fusion peptide of antigenic epitope of avian infectious bronchitis virus and its application in indirect ELISA detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410710444.8A CN118546258B (en) | 2024-06-03 | 2024-06-03 | A fusion peptide of antigenic epitope of avian infectious bronchitis virus and its application in indirect ELISA detection |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411573590.7A Division CN119320459B (en) | 2024-06-03 | 2024-06-03 | Avian infectious bronchitis virus epitope fusion peptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118546258A CN118546258A (en) | 2024-08-27 |
| CN118546258B true CN118546258B (en) | 2024-11-29 |
Family
ID=92449704
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410710444.8A Active CN118546258B (en) | 2024-06-03 | 2024-06-03 | A fusion peptide of antigenic epitope of avian infectious bronchitis virus and its application in indirect ELISA detection |
| CN202411573590.7A Active CN119320459B (en) | 2024-06-03 | 2024-06-03 | Avian infectious bronchitis virus epitope fusion peptide |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411573590.7A Active CN119320459B (en) | 2024-06-03 | 2024-06-03 | Avian infectious bronchitis virus epitope fusion peptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN118546258B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110376385A (en) * | 2019-07-25 | 2019-10-25 | 扬州大学 | A kind of preparation method and application of gene engineering expression QX type avian infectious bronchitis virus S1 proteantigen |
Family Cites Families (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0221609A1 (en) * | 1985-10-31 | 1987-05-13 | Duphar International Research B.V | New antigenically active proteins and peptides, and infectious bronchitis virus (IBV) vaccines |
| DE69020848D1 (en) * | 1989-10-20 | 1995-08-17 | Akzo Nobel Nv | IBV vaccine. |
| WO2006009011A1 (en) * | 2004-07-15 | 2006-01-26 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Coronaviral spike s1 fused protein and expression vector therefor |
| CN1763085A (en) * | 2005-11-02 | 2006-04-26 | 浙江大学 | Chicken infectious bronchitis virus S1 recombinant protein antigen, preparation method and use |
| AR072976A1 (en) * | 2008-08-08 | 2010-10-06 | Wyeth Corp | ISOLATED INFECTIOUS BRONCHITIS (IB) VIRUSES, VACCINE COMPOSITION AND METHOD TO PREPARE AN INFECTIOUS BRONCHITIS (IB) VIRUS ATTACHED LIVE |
| CN102851257A (en) * | 2012-08-27 | 2013-01-02 | 上海启盛生物科技有限公司 | Attenuated vaccine strain for avian infectious bronchitis virus and application thereof |
| WO2014077096A1 (en) * | 2012-11-15 | 2014-05-22 | 一般財団法人化学及血清療法研究所 | Method for preventing infections using combination of vector vaccine and live vaccine |
| US20140141043A1 (en) * | 2012-11-16 | 2014-05-22 | Auburn University | Compositions, vectors, kits, and methods for immunizing against avian infectious bronchitis virus |
| CN105092839A (en) * | 2015-08-20 | 2015-11-25 | 中国农业科学院哈尔滨兽医研究所 | Detection kit for chicken infectious bronchitis indirect ELISA antibody |
| CN105949321B (en) * | 2016-05-04 | 2019-08-23 | 中国农业科学院上海兽医研究所 | The B cell antigen epi-position polypeptide and its vaccine of infectious bronchitis virus separation strains S1 albumen |
| DE102016215118A1 (en) * | 2016-08-12 | 2018-02-15 | Anicon Labor Gmbh | Infectious Bronchitis (IB) Virus Variants and Related Compositions, Uses and Methods |
| WO2018112051A1 (en) * | 2016-12-14 | 2018-06-21 | Merial, Inc. | Recombinant hvt vectors expressing multiple antigens of avian pathogens, and vaccines comprising them |
| US10772953B2 (en) * | 2017-08-09 | 2020-09-15 | Auburn University | Recombinant spike ectodomain proteins, compositions, vectors, kits, and methods for immunizing against avian infectious bronchitis virus |
| UA129674C2 (en) * | 2018-10-31 | 2025-07-02 | Бьорінгер Інгельхайм Ветмедіка Гмбх | VACCINE AGAINST INFECTIOUS BRONCHITIS VIRUS 4/91 WITH HETEROLOGICAL SPIKE PROTEIN |
| KR102302141B1 (en) * | 2018-12-13 | 2021-09-16 | 주식회사 메디안디노스틱 | Recombinant nucleocapsid for detecting the antibodies against Infectious Bronchitis Virus and using the same |
| CN111254221A (en) * | 2020-02-24 | 2020-06-09 | 中国动物卫生与流行病学中心 | Method for detecting four main avian respiratory syndrome viruses |
| US20240374711A1 (en) * | 2021-06-11 | 2024-11-14 | Auburn University | RECOMBINANT NEWCASTLE DISEASE VIRUS (rNDV) VECTORS AND METHODS OF USING THE SAME |
| CN115819524A (en) * | 2022-11-30 | 2023-03-21 | 山东畜牧兽医职业学院 | Polypeptide for detecting infectious bronchitis virus antibody and detection kit thereof |
| CN116375819A (en) * | 2023-01-12 | 2023-07-04 | 乾元浩生物股份有限公司 | ELISA detection kit for chicken infectious bronchitis S protein antibody |
| CN116298278A (en) * | 2023-04-18 | 2023-06-23 | 浙江大学 | A kind of ELISA kit and application for the detection of infectious bronchitis virus neutralizing antibody |
| CN117430675A (en) * | 2023-09-27 | 2024-01-23 | 华南农业大学 | Antigen of kit for detecting infectious bronchitis in chickens, preparation method of antigen and kit |
-
2024
- 2024-06-03 CN CN202410710444.8A patent/CN118546258B/en active Active
- 2024-06-03 CN CN202411573590.7A patent/CN119320459B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110376385A (en) * | 2019-07-25 | 2019-10-25 | 扬州大学 | A kind of preparation method and application of gene engineering expression QX type avian infectious bronchitis virus S1 proteantigen |
Also Published As
| Publication number | Publication date |
|---|---|
| CN119320459A (en) | 2025-01-17 |
| CN118546258A (en) | 2024-08-27 |
| CN119320459B (en) | 2025-04-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115043948B (en) | Monkey pox virus specific fusion protein antigen and preparation method and application thereof | |
| AU2020102599A4 (en) | Indirect ELISA Detection Method and Application of DHAV-3 Antibody Based on VP2 or VP4 Recombinant Protein Antigen | |
| CN116715737B (en) | Novel target antigen of coronavirus receptor binding domain B cell epitope and expression and application thereof in Peg bacterium Mao Zhan | |
| CN113956362B (en) | Recombinant feline parvovirus VP2 protein antigen and application thereof in antibody diagnosis and vaccine preparation | |
| KR102707584B1 (en) | Recombinant nucleocapsid protein for diagnosis and vaccine of COVID-19 and use thereof | |
| CN112500494B (en) | Antigen for detecting novel coronavirus and preparation method thereof | |
| CN110208518A (en) | Indirect ELISA detection method and its detection kit based on 3 type adenovirus antibody of Fiber1 Protein Detection duck | |
| CN113637056A (en) | A kind of test kit for identifying Brucella bovis and other species of Brucella | |
| CN118666971B (en) | Conserved B cell epitopes of VP1 protein of chicken infectious anemia virus capsid and their application in functional display and detection of Peg fimbriae | |
| CN110554187B (en) | Expression protein for detecting bovine viral diarrhea virus antibody, ELISA kit, preparation method and application thereof | |
| CN113203854B (en) | Indirect ELISA kit for detecting cat coronavirus antibody | |
| CN118546258B (en) | A fusion peptide of antigenic epitope of avian infectious bronchitis virus and its application in indirect ELISA detection | |
| CN108680741B (en) | Chicken infectious bronchitis virus 5b ELISA antibody detection kit and its application | |
| CN102618557A (en) | Recombinant avian flavivirus E protein and application thereof | |
| CN105693831A (en) | Livestock S2 vaccine immunity and Brucella melitensis/abortus infection IELISA (enzyme linked immunosorbent assay) detection reagent | |
| CN110376385B (en) | A kind of preparation method and application of genetically engineered expression of QX type chicken infectious bronchitis virus S1 protein antigen | |
| CN116284434B (en) | A variant pseudorabies virus gC-gD epitope concatemer and its application | |
| CN119119295B (en) | Mycoplasma hyopneumoniae fusion protein and preparation method and application thereof | |
| CN114751964B (en) | Universal vaccine protein fragment of beta coronavirus, screening method and application thereof | |
| KR102717226B1 (en) | Vaccine composition, composition for diva and method for diva using thereof | |
| CN113564192B (en) | Tick-borne encephalitis virus capsid protein C prokaryotic expression vector, recombinant strain and application thereof | |
| CN120685906A (en) | Application of recombinant VP1 protein as coating antigen in preparation of ELISA (enzyme-Linked immunosorbent assay) detection kit for avian encephalomyelitis virus antibody | |
| CN115716867A (en) | A V-type secretion system MisL exhibits and expresses the novel coronavirus receptor binding domain B cell epitope antigen and its application | |
| CN119930766A (en) | A type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein and its application | |
| CN118067988A (en) | Application of streptococcus suis antigen protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |