A kind of chicken infectious bronchitis virus antigen epitope fusion peptide

Abstract

The present invention provides an antigenic epitope fusion peptide of infectious bronchitis virus of chickens. 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: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

[0001] This invention is a divisional application of the Chinese invention patent with application number "202410710444.8" and invention name "A chicken infectious bronchitis virus antigen epitope fusion peptide and its application in indirect ELISA detection". The application date of the parent case is June 3, 2024. Technical Field

[0002] The invention belongs to the technical field of vaccine antigen preparation, and in particular relates to an avian infectious bronchitis virus antigen epitope fusion peptide. Background Art

[0003] Infectious bronchitis (IB) is an acute, highly contagious, viral respiratory disease caused by infectious bronchitis virus (IBV). Chickens of all ages and breeds are susceptible to infectious bronchitis, especially chicks aged 1 to 4 weeks, with a mortality rate of 40% to 90%. Although vaccines are widely used for prevention, IB has not been effectively controlled, and mutants or new genotypes continue to emerge. At present, IB has occurred and spread in most parts of the world, but the genotypes or strains prevalent in different regions are different. So far, there are at least three genotypes in my country, namely GI-19, GVI and GVII. Among them, GI-19 type, also known as QX type, was first discovered in my country and is the most prevalent IBV genotype in China, causing huge losses to my country's poultry industry.

[0004] IBV is a single-stranded positive-strand RNA virus with a genome of approximately 27.6 kb, belonging to the genus gamma coronavirus. The virus particle is spherical with a diameter of 82 nm; the virus particle is composed of four structural proteins: spike (S) protein, membrane (M) glycoprotein, nucleocapsid (N) protein and small membrane (E) protein.

[0005] At present, the laboratory methods used for IB antibody detection mainly include enzyme-linked immunosorbent assay (ELISA), agar diffusion precipitation test (AGP) and virus neutralization test (VN). All three detection methods have good specificity, but AGP has poor sensitivity; neutralization test is complicated, time-consuming and requires high laboratory conditions; ELISA method is suitable for laboratory serological diagnosis and large-scale epidemic investigation because of its fast speed, low requirements for experimental conditions, no need for aseptic operation, and the ability to detect a large number of samples in a short time. Summary of the invention

[0006] The invention provides an avian infectious bronchitis virus antigen epitope fusion peptide and application thereof in indirect ELISA detection. The prepared antigen epitope fusion peptide can be used for preparing antibodies and can also be used as a coating antigen for preparing indirect ELISA detection products.

[0007] The chicken infectious bronchitis virus antigen epitope fusion peptide provided by the present invention is the antigen epitope fusion peptide S1-I, whose amino acid sequence is SEQ ID NO: 2; and its corresponding encoding nucleotide sequence is SEQ ID NO: 1;

[0008] The chicken infectious bronchitis virus antigen epitope fusion peptide provided by the present invention is the antigen epitope fusion peptide S1-II, and its amino acid sequence is SEQ ID NO:4; its corresponding encoding nucleotide sequence is SEQ ID NO:3;

[0009] Codon optimization was performed with reference to the Escherichia coli codon preference table. The nucleotide sequence of S1-I after optimization was SEQ ID NO: 5, and the nucleotide sequence of S1-II was SEQ ID NO: 6.

[0010] The present invention also provides a recombinant expression vector, in which a nucleic acid fragment encoding the antigen epitope fusion peptide is inserted;

[0011] In another aspect, the present invention further provides a prokaryotic expression host bacterium, wherein the prokaryotic expression host bacterium carries the above-mentioned recombinant expression vector;

[0012] The present invention also provides the use of the chicken infectious bronchitis virus antigen epitope fusion peptide as a coating antigen;

[0013] As a specific record of the embodiment, the application is used to prepare an indirect ELISA detection product;

[0014] Another aspect of the present invention also provides an indirect ELISA detection product, wherein the coating antigen is the above-mentioned chicken infectious bronchitis virus antigen epitope fusion peptide;

[0015] Furthermore, the antigen coating concentration is 0.5-1 μg / mL.

[0016] Another aspect of the present invention provides an indirect ELISA detection method for avian infectious bronchitis virus, which uses the above-mentioned indirect ELISA detection product for detection, wherein the S1-I antigen coating concentration is 1 μg / mL, the serum dilution is 1:1000, the secondary antibody dilution is 1:10000, and 5% skim milk is the best blocking solution.

[0017] The recombinant protein S1-II was used as the coating antigen, the antigen coating concentration was 0.5 μg / mL, the serum dilution was 1:1000, the secondary antibody dilution was 1:5000, and 5% skim milk was the best blocking solution.

[0018] The present invention uses bioinformatics software to analyze the antigenic epitopes of the S1 protein of the infectious bronchitis virus strain, and then fuses multiple antigenic epitopes to obtain an 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Schematic diagram of the sequence structure of antigen epitope polypeptides S1-I and S1-II,

[0020] Figure 2 :S1-I Sequence analysis results,

[0021] Figure 3 :S1-II sequence analysis results;

[0022] Figure 4 : Prokaryotic expression vector double enzyme digestion nucleic acid gel map, where M is the nucleic acid relative molecular mass standard (5000bp); lanes 1-2 are pET-30a (+) -S1-I double enzyme digestion products; lanes 3-4 are pET-30a (+) -S1-II double enzyme digestion products;

[0023] Figure 5 : Recombinant protein S1-I / II expression purification electrophoresis detection diagram, wherein Figure A is the recombinant protein S1-I expression purification electrophoresis detection diagram; Figure B is the recombinant protein S1-II expression purification electrophoresis detection diagram; in the figure, M is the protein relative molecular mass standard (180kDa), lane 1 is the concentrated recombinant protein S1-I; lane 2 is the concentrated recombinant protein S1-II;

[0024] Figure 6 : The positive serum binding to the strain after recombinant protein immunization, wherein Figure A is the S1-I positive serum binding to the strain, Figure B is the S1-II positive serum binding to the strain, and M in the figure is the protein relative molecular mass standard (180kDa); Lane 1 is the GI-19 type IBV strain sample; Lane 2 is the GI-19 type IBV strain sample;

[0025] Figure 7 :Identification diagram of the neutralization level produced by the body stimulated by the recombinant protein S1-I / II;

[0026] Figure 8 : The judgment diagram of the indirect ELISA positive value, wherein Figure A is the judgment diagram of the indirect ELISA positive value when the recombinant protein S1-I is the coating antigen, and Figure B is the judgment diagram of the indirect ELISA positive value when the recombinant protein S1-II is the coating antigen; DETAILED DESCRIPTION

[0027] The invention uses various bioinformatics to analyze the antigenicity of the S1 protein of IBV, designs and selects the dominant antigen epitope on the S1 gene, performs codon optimization and synthesis, constructs a prokaryotic expression plasmid, and then transforms the plasmid into competent cells such as E. coli Rosetta and E. coli BL21 (DE3) for prokaryotic expression and purifies the recombinant protein. The purified protein is further coated on an ELISA plate as an antigen, and IBV (GVI-1), IBV (H120), IBV (ahysx-1), AIV (H5), AIV (H9), NDV, and GAstV positive sera are respectively detected by enzyme-linked immunosorbent assay (ELISA). The S1-I recombinant protein with the best reaction effect with QX type positive sera and weaker reaction effect with other non-GI-19 type positive sera is selected as the detection antigen, and an ELISA method is established for the detection of GI-19 type IB vaccine immune antibodies and clinical GI-19 type IBV infection monitoring.

[0028] The information of plasmids, strains, and serum in the examples of the present invention are as follows:

[0029] After codon optimization, the S1-I and S1-II sequences were sent to Qingdao Ruibo Kexing Biotechnology Co., Ltd. for synthesis; E. coli Rosetta and E. coli BL21 (DE3) competent cells were purchased from Beijing Bomade Gene Technology Co., Ltd.; IBV (GI-19), IBV (GVI-1), IBV (H120), IBV (ahysx-1) AIV (H5), AIV (H9), NDV, GAstV positive sera and negative sera were all stored in the laboratory.

[0030] IPTG solution, ELISA coating solution, stop solution, and two-component TMB colorimetric solution were purchased from Beijing Solebow Technology Co., Ltd.; horseradish perhydrolase (HRP)-labeled sheep anti-chicken IgG was purchased from Beijing Bioson Biotechnology Co., Ltd.; plasmid minipreparation kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; 96-well detachable ELISA plate was purchased from Wuxi Nice Life Science Co., Ltd., and primer synthesis and sequencing were completed by Qingdao Ruibo Kexing Biotechnology Co., Ltd.

[0031] The present invention is described in detail below in conjunction with embodiments and drawings.

[0032] Example 1: Screening and sequence optimization of antigen epitope polypeptides

[0033] The online tool http: / / imed.med.ucm.es / Tools / antigenic.pl was used to predict and analyze the antigenic epitopes on the S1 gene fragment of the IBV GI-19 strain (GenBank accession number MN548289) (Table 1). The epitope sequences were separated by alanine-alanine-tyrosine (AAY) linker sequences to construct two antigenic peptide epitopes S1-I and S1-II with strong antigenicity, good hydrophilicity and flexibility ( Figure 1 ), DNAStar Protean software was used to analyze the secondary structure, antigenicity index, hydrophilicity, and flexibility of the multi-epitope peptides. The secondary structure, antigenicity index, hydrophilicity, and flexibility of S1-I and S1-III are shown in Figure 2. Figure 2 and Figure 3 As shown, the two polypeptide segments have good antigenicity.

[0034] Table 1: IBV S1 antigen epitope prediction table

[0035]

[0036] The nucleotide sequence corresponding to S1-1 is as follows (SEQ ID NO: 1):

[0037] AAGTCACTGTTTTTAGTGACCATTTTGTGTGCACTATGTAGTGCAAATGCTGCTTACAATAATTATGTGTACTACTACCAAAGTGCTGCTGCTTACCTACAAGGAGGTGCTTATGCAGTAGTCAATGCTGCTTACTCTGCACATCAGTGCACTGTTGGTGTTATTAAGGATGTTTATAATCAAAGTGTGGCTTCCATAGCTATGACAGCACCTCTTGCTGCTTACTCTAAGTCACAATTCTGTAGTGCACACTGTAATTTTTCTGAAATTACAGTTTTTGTCACACATTGTTATAGTGCTGCTTACGGTAGTGGGTCTTGTCCTATAACAGCTGCTTACTTATTTTATAATTTAACAGTTAGCGTATCTAAAGCTGCTTACAAATCTTTTCAATGTGTTAACAACTTCACATCTGTTTATTTAAATGGTGATCTTGTTTTTACTGCTGCTTACGATGTTACGTCAGCAGGTGTGTATTTTAAAGCAGCTGCTTACAAAGAATTTAAGGTTCTTGCTTACTTTGTTAATGCTGCTTACGCACAAGATGTAGTTTTGTGCGACGCTGCTTACCCCAAGGGTTTGCTAGCTTGTCAAGCTGCTTACACTAATAGTACTTTGGTTAGGGAAAAGTTCATCGTCTATCGCGAAGCTGCTTACGTTAATACTACTCTGGCGTTAACTGCTGCTTACGTTAATACTTTTCATTTATACCAAACAGCTGCTTACTATAATTTTAATTTGTCATTTCTGAGTCAGTTTGTGTATAAG;

[0038] The amino acid sequence corresponding to S1-I is as follows (SEQ ID NO: 2):

[0039] KSLFLVTILCALCSANAAYNNYVYYYQSAAAYLQGGAYAVVNAAYSAHQCTVGVIKDVYNQSVASIAMTAPLAAYSKSQFCSAHCNFSEITVFVTHCYSAAYGSGSCPITAAYLFYNLTVSVSKAAYKSFQCVNNFTSVYLNGDLVFTAAYDVTSAGVYFKAAAYKEFKVLAYFVNAAYAQDVVLCDAAYPKGLLACQAAYTNSTLVREKFIVYREAAYVNTTLALTAAYVNTFHLYQTAAYYNFNLSFLSQFVYK;

[0040] The nucleotide sequence corresponding to S1-II is as follows (SEQ ID NO: 3):

[0041] 。

[0042] The amino acid sequence corresponding to S1-II is as follows (SEQ ID NO: 4):

[0043] SAHQCTVGVIKDVYNQSVASIAMTAPLAAYSKSQFCSAHCNFSEITVFVTHCYSAAYGSGSCPITAAYLFYNLTVSVSKAAYKSFQCVNNFTSVYLNGDLVFTAAYDVTSAGVYFKAAAYKEFKVLAYFVNAAYAQDVVLCDAAYPKGLLACQAAYTNSTLVREK FIVYREAAYVNTTLALTAAYAAYVNTFHLYQTAAYYNFNLSFLSQFVYKAAYYGSYHPSCSFAAYWFNSLSSVSLTYGPLQGGCKQSVFSGKATCCYAYSYAAYGPMACKGVYSGAAYFECGLLVYVTAAYTEPLVLTQAAYGAIDVFVVQGIYGLNYYKVNPCED.

[0044] Codon optimization was performed with reference to the Escherichia coli codon preference table. The nucleotide sequence of S1-I after optimization is as follows (SEQ ID NO: 5):

[0045] AAAAGCCTGTTCCTGGTTACCATCCTGTGCGCCCTGTGTTCTGCCAACGCGGCATACAATAACTACGTGTACTACTACCAGAGCGCGGCTGCTTACCTGCAAGGTGGCGCTTACGCTGTCGTAAACGCTGCATACTCCGCTCATCAGTGCACCGTTGGTGTAATTAAGGATGTTTACAACCAGTCCGTAGCATCCATTGCGATGACCGCGCCGCTGGCTGCATATTCTAAGTCCCAGTTCTGCTCCGCGCACTGCAATTTCAGCGAGATTACTGTTTTCGTAACCCACTG CTACTCCGCAGCGTATGGCTCTGGTAGCTGCCCGATCACTGCTGCCTACCTGTTCTATAACCTGACCGTGAGCGTGTCTAAAGCCGCGTATAAATCTTTCCAGTGCGTGAACAACTTCACCTCCGTTTACCTGAACGGTGACCTGGTTTTTACTGCCGCCTACGATGTAACCTCTGCCGGTGTTTATTTCAAGGCAGCGGCGTACAAAGAATTCAAGGTGCTGGCGTACTTCGTCAACGCCGCTTATGCCCAGGATGTTGTGCTGTGTGACGCAGCGTACCCGAAAGGTCTGCTGGCGTGTCAGGCAGCGTACACGAATTCTACCCTGGTACGCGAGAAATTCATTGTCTACCGTGAGGCTGCTTACGTCAACACCACTCTGGCACTGACCGCAGCCTACGTGAACACCTTCCATCTGTATCAGACTGCGGCGTACTACAACTTCAACCTGTCCTTTCTGAGCCAGTTCGTGTACAAA;

[0046] The nucleotide sequence of the optimized S1-II is as follows (SEQ ID NO: 6):

[0047] TCTGCCCACCAGTGCACCGTTGGCGTGATTAAAGACGTTTACAACCAGTCCGTTGCTAGCATTGCAATGACTGCCCCGCTGGCAGCCTACTCCAAAAGCCAGTTTTGTTCCGCGCACTGCAACTTCTCTGAGATCACCGTATTTGTTACCCATTGTTATTCTGCGGCCTATGGCTCTGGTAGCTGTCCGATCACGGCGGCGTATCTGTTTTATAACCTGACCGTCAGCGTTTCCAAAGCGGCTTACAAGTCCTTCCAGTGCGTCAACAATTTCACCTCTGTTTATCTGAACGGTGACCTGGTGTTTACTGCGGCATATGACGTAACCAGCGCTGGCGTTTATTTTAAAGCGGCGGCGTACAAAGAATTCAAAGTACTGGCATACTTCGTCAACGCAGCGTACGCTCAGGATGTAGTTCTGTGCGACGCAGCATACCCGAAAGGTCTGCTGGCTTGTCAGGCTGCGTACACTAACTCCACCCTGGTTCGCGAAAAGTTCATCGTATACCGCGAAGCGGCTTACGTAAACACGACTCTGGCACTGACCGCAGCTTACGCTGCTTACGTAAACACGTTCCACCTGTACCAAACCGCGGCCTACTACAACTTCAATCTGAGCTTTCTGAGCCAGTTCGTATACAAGGCTGCCTACTACGGCTCCTACCACCCGTCTTGCTCTTTCGCTGCATACTGGTTCAACAGCCTGTCTGTCTCCCTGACCTACGGCCCGCTGCAGGGTGGCTGCAAACAGAGCGTCTTCTCCGGTAAAGCGACCTGCTGTTATGCATACTCCTATGCAGCGTATGGTCCGATGGCGTGTAAAGGTGTTTACTCCGGCGCCGCCTACTTTGAGTGCGGTCTGCTGGTATATGTCACCGCGGCTTATACTGAACCTCTGGTCCTGACCCAGGCGGCATACGGCGCTATTGACGTATTCGTTGTTCAGGGCATCTATGGCCTGAACTATTACAAGGTGAACCCGTGTGAAGAT。

[0048] Example 2: Construction of prokaryotic expression vector and recombinant expression of antigen epitope polypeptide

[0049] 1. Construction of recombinant expression plasmid vector

[0050] Two pairs of primers were designed and synthesized according to the optimized sequence. The specific sequence information is as follows:

[0051] S1-IF:5′-AA GAATTC AAAAGCCTGTTCCTGGTTAC-3′,

[0052] S1-IR:5′-TT CTCGAG TTTGTACACGAACTGGCTCA-3′;

[0053] S1-II-F:5′-AA GAATTC TCTGCCCACCAGTGCACCGT-3′,

[0054] S1-II-R:5′-TT CTCGAG ATCTTCACACGGGTTCACCT-3′ (the underlined parts indicate the EcoRI and XhoI restriction sites introduced respectively), the theoretical length of S1-I is 768 bp, and S1-II is 990 bp. The plasmid containing the genome of S1-I and S1-II was extracted as a template and used Max DNA Polymerase kit was used for PCR amplification. The reaction system was: 2*Buffer 10μL, upstream and downstream primers 1μL each, plasmid 1μL, RNase free H2O 7μL. The reaction conditions were: 98℃2min, 98℃10s, 52℃15s, 72℃1min, 35 cycles; 72℃10min; 4℃ to terminate the reaction. After agarose gel electrophoresis, the PCR amplification product was purified by gel recovery kit, and the target gene fragment and pET30a(+) vector were double-digested with EcoRI and XhoI restriction endonucleases and recovered and purified. T4DNA ligase was connected at 16℃ overnight, and the ligation product was transformed into DH5α competent cells. Plasmid DNA was extracted using a plasmid extraction kit. After being identified as correct by EcoRI and XhoI double digestion, it was sent to Qingdao Ruibo Kexing Biotechnology Co., Ltd. for sequencing and identification.

[0055] The designed restriction endonucleases EcoRI and XhoI were used to double-digest the recombinant prokaryotic expression plasmids pET-30a(+)-S1-I and pET-30a(+)-S1-II to identify the target gene. After double digestion, the lengths of S1-I, S1-II, and S1-II were 768 bp, 990 bp, and 5400 bp, respectively. The sizes of the fragments and vectors were consistent with the target fragment and the insertion vector ( Figure 4 ).

[0056] 2. Gene induction expression and purification and concentration of expression products

[0057] The plasmid was extracted from the bacterial solution containing pET-30a(+)-S1-I and pET-30a(+)-S1-II, and transferred into E.coli Rosetta and E.coli BL21(DE3) competent cells. 50μL was spread on LB agar plates containing kan (100μg / mL), cultured at 37℃ overnight, and a single white colony was picked and cultured in LB liquid medium containing kan overnight. Then the culture was expanded at a ratio of 1:100, and when the OD value reached about 0.4-0.6, IPTG was added to a final concentration of 0.8mmol / L, and cultured at 28℃ for 12h. The bacterial cells were broken by ultrasonic wave, and the broken bacterial solution was centrifuged at 4℃, 10000rmp / min for 10min, and the supernatant and precipitate of the lysate were separated. The expression form of the recombinant protein was analyzed by SDS-PAGE. The inclusion body was washed to remove impurities under denaturing conditions, and the denatured protein was dialyzed at different urea concentrations for gradual renaturation. The purified protein was detected by SDS-PAGE and Western blot, and the concentration of the purified protein was determined by a nucleic acid protein analyzer. TM Affinity chromatography column was used to purify a large amount of induced expressed proteins. The purification effect was analyzed by SDS-PAGE, and the purified proteins were named S1-I and S1-II. The purified proteins were placed in a dialysis bag, and the dialysis bag containing the recombinant protein was placed in the protein dialysis solution for renaturation. The protein dialysis solution containing different concentrations of urea (8mmol / L, 6mmol / L, 4mmol / L, 2mmol / L, 1mmol / L) was dialyzed in a gradient from high to low concentration, replaced in the next gradient protein dialysis solution at intervals of 12h, and finally dialyzed in PBS for 12h. The dialysis bag was embedded with sucrose for protein concentration, and the renatured purified protein was identified by Western-blot and SDS-PAGE.

[0058] The recombinant proteins S1-I and S1-II that were induced to express in large quantities were concentrated by urea gradient dialysis. The refolded purified proteins were identified by Western blot and SDS-PAGE. The results showed that relatively pure recombinant proteins were obtained, and the recombinant proteins could react with GI-19 IBV positive serum to produce specific bands ( Figure 5 ).

[0059] Example 3: Recombinant proteins S1-I and S1-II as coating antigens

[0060] 1. Preparation of GI-19 positive serum

[0061] SPF chickens were first immunized at 14 days of age with white oil as adjuvant, and 100 ng of recombinant protein S1-I / II was injected into the leg muscle. Two weeks later, the same immunization dose was used for two booster immunizations, and serum was collected every week. The GI-19 IBV strain stored in the laboratory was diluted to 10 3 EID 50 / 0.1ml for Western-blot sample preparation, and preliminary determination of antibody production in the body by Western-blot. Western-blot results show that antibodies can bind to strain samples. Virus neutralization experiments are performed to determine the level of neutralizing antibodies in serum. The strain is diluted to 200EID with PBS. 50 / 0.1mL, mixed with the collected serum, neutralized at room temperature for 1 hour, inoculated into the allantoic cavity with 10-11 day old SPF chicken embryos, 0.2mL per embryo, placed at 37℃ and observed for 144 hours. Chicken blood with a neutralizing antibody level of 6Log2 was collected by heart blood sampling, incubated at 37℃ for 1 hour, incubated at 4℃ for 1 hour, centrifuged at 4000rmp / min for 10 minutes, and the collected serum was packaged and stored at -20℃ for later use.

[0062] The GI-19 IBV strain stored in the laboratory was diluted to 10 3 EID 50 / 0.1ml for Western-blot sample preparation, and the positive serum was diluted to 1:200, and the Western-blot method was used for detection. The Western-blot results showed that the recombinant protein S1-I / II injected into SPF chickens could stimulate the body to produce antibodies ( Figure 6 The results of neutralizing antibody test showed that the neutralizing antibody level of S1-I serum could reach up to 6Log2(3 / 5), and the antibody levels of the remaining SPF chickens were 5Log2(1 / 5) and 4Log2(1 / 5). The neutralizing antibody level of S1-II serum could reach up to 6Log2(2 / 5), and the neutralizing antibody levels of the remaining SPF chickens were 5Log2((2 / 5), 4Log2(1 / 5)( Figure 7 ).

[0063] 2. Establishment of indirect ELISA detection method

[0064] 1) Optimization of antigen coating concentration, primary antibody serum, and enzyme-labeled antibody dilution

[0065] The coating concentration of the recombinantly expressed S1-I and S1-II epitope polypeptides and the dilution of the primary antiserum were determined by the chessboard titration method. The purified S1-I and S1-II epitope polypeptides were coated on the ELISA plate at 4°C and 37°C for 1h at 4μg / L, 1μg / mL, 0.5μg / mL, and 0.25μg / mL. After washing three times with 1×PBST, 5% skim milk was blocked at 37°C for 1h. After washing three times with PBST, the positive serum and negative serum were added to the ELISA plate at 8 dilutions of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000, and reacted at 37°C for 1h. Wash the plate with 1×PBST, add HRP-labeled rabbit anti-chicken IgY antibody (1:5000 dilution, 1:10000 dilution) and incubate at 37°C for 1 hour. After washing three times with 1×PBST, develop the color with 100μL TMB solution. Terminate the reaction with 50μL ELISA stop solution. Measure the absorbance at OD450 using an enzyme reader. Determine the optimal serum dilution and antigen coating concentration based on the maximum P / N value.

[0066] The recombinant protein S1-I was used as the coating antigen, and the positive and negative sera were tested by chessboard titration. The results showed (Table 2) that when the antigen coating concentration was 1 μg / mL, the serum dilution was 1:1000, and the secondary antibody dilution was 1:10000, the P / N value was 28.178, which was the maximum. Therefore, this condition was selected as the reaction condition for subsequent experiments.

[0067] Table 2: Determination of recombinant protein S1-I coating concentration, serum dilution and optimal enzyme-labeled secondary antibody dilution

[0068]

[0069] The recombinant protein S1-II was used as the coating antigen, and the positive and negative sera were tested by chessboard titration. The results showed (Table 3) that 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 P / N value was 14.917, which was the maximum. Therefore, this condition was selected as the reaction condition for subsequent experiments.

[0070] Table 3: Determination of recombinant protein S1-II coating concentration, serum dilution and optimal enzyme-labeled secondary antibody dilution

[0071]

[0072] 2) Determine the ELISA blocking solution

[0073] The ELISA plates coated with S1-I and S1-II were blocked with 1% BSA, 2% BSA, 5% skim milk and 10% skim milk, respectively, at 37°C for 1h. After washing three times with 1×PBST, the positive serum and negative serum at the optimal dilution were used as primary antibodies and reacted at 37°C for 1h. After washing three times with 1×PBST, the HRP-labeled rabbit anti-chicken IgY antibody was incubated at 1:10000 at 37°C for 1h. After washing three times with 1×PBST, 100μL TMB substrate colorimetric solution was added and 50μL ELISA stop solution was used to stop the reaction. The absorbance was measured at OD450 using a universal microplate reader. According to the maximum value of P / N, the optimal ELISA blocking solution and enzyme-labeled antibody dilution were determined.

[0074] The results of ELISA test with recombinant protein S1-I as coating antigen and 1% BSA, 2% BSA, 5% skim milk and 10% skim milk as blocking solution (Table 4) show that when 10% skim milk is used to block the ELISA plate, the P / N value is 33.844, which is the largest. Therefore, 5% skim milk is determined to be the best blocking solution.

[0075] Table 4: Determination of blocking conditions for recombinant protein S1-I as coating antigen

[0076]

[0077] The results of ELISA test with recombinant protein S1-II as coating antigen and 1% BSA, 2% BSA, 5% skim milk and 10% skim milk as blocking solution (Table 5) show that when 10% skim milk is used to block the ELISA plate, the P / N value is the largest at 13.015. Therefore, 5% skim milk is determined to be the best blocking solution.

[0078] Table 5: Determination of blocking conditions for recombinant protein S1-II as coating antigen

[0079]

[0080] 3) Determination of reaction time of primary and secondary antibodies

[0081] The ELISA plate coated with S1-I / II was blocked with 10% skim milk at 37°C for 1h. After washing three times with 1×PBST, the positive serum and negative serum at the optimal dilution were used as primary antibodies and incubated at 37°C for 0.5, 1h, 1.5h and 2h, respectively. After washing three times with 1×PBST, the HRP-labeled rabbit anti-chicken IgY antibody was diluted at a ratio of 1:10000 and incubated at 37°C for 0.5h, 1h, 1.5h and 2h, respectively. After washing three times with 1×PBST, 100μL TMB substrate colorimetric solution was added and 50μL ELISA stop solution was used to stop the reaction. The absorbance was measured at OD450 using an ELISA reader. The reaction time of the primary antibody serum and the enzyme-labeled secondary antibody was determined according to the maximum value of P / N.

[0082] Determination of reaction time of primary antiserum and enzyme-labeled antibody

[0083] With the recombinant protein S1-I as the coating antigen, when the primary antibody serum was incubated for 1 h and the enzyme-labeled antibody reaction time was 1 h, the P / N value was 31.857, which was the maximum (Table 6). Therefore, the optimal incubation time of the primary antibody and the enzyme-labeled antibody reaction time were determined to be 1 h.

[0084] Table 6: Determination of blocking conditions for recombinant protein S1-I as coating antigen

[0085]

[0086] With the recombinant protein S1-II as the coating antigen, when the primary antibody serum was incubated for 0.5 h and the enzyme-labeled antibody reaction time was 1 h, the P / N value was 16.948, which was the maximum (Table 7). Therefore, the optimal incubation time of the primary antibody and the enzyme-labeled antibody reaction time were determined to be 1 h.

[0087] Table 7: Determination of blocking conditions for recombinant protein S1-II as coating antigen

[0088]

[0089] 4) Indirect ELISA positive and negative determination criteria

[0090] Select 24 chicken sera, set up positive controls, and determine the OD of negative and positive sera according to the optimized indirect ELISA detection method. 450nm The mean (X) and standard deviation (SD) of the N / P values ​​were calculated. S / P ≥ X + 3SD was considered positive, and S / P < X + 3SD was considered negative.

[0091] Using recombinant protein S1-I as the coating antigen, the OD values ​​of 24 sera at a dilution of 1:2000 were counted. 450 nm value, calculate the OD of all negative serum 450The X of nm is 0.064, S is 0.011, which is converted to the critical value of negative and positive: S / P = 0.097. When S / P ≥ 0.097, it is judged as positive, and when S / P is less than 0.097, it is judged as negative. Figure 8 A). Using recombinant protein S1-II as the coating antigen, the OD450 nm values ​​of 24 sera at a dilution of 1:2000 were counted, and the OD450nm values ​​of all negative sera were calculated as X=0.070, S=0.022, which were converted to the critical value for determining negative and positive: S / P=0.136. When S / P≥0.136, it was determined to be positive, and when S / P was less than 0.136, it was determined to be negative ( Figure 8 B) The experimental results show that the detection limit is lower when the recombinant protein S1-I is used as the coating antigen.

[0092] 5) Repeatability test

[0093] The intra-batch repeatability test was performed using 3 batches of ELISA plates coated with the same batch of antigens; the inter-batch repeatability test was performed using 3 batches of ELISA plates coated with antigens in different batches. Each time, 6 serum samples were used for indirect ELISA test, and 3 replicates were set for each serum sample. The OD was read. 450nm The values ​​were calculated, X, S and coefficient of variation (CV) were calculated, and the results were statistically analyzed.

[0094] The results of intra-batch and inter-batch repeatability tests using recombinant protein S1-I as the coating antigen showed that the minimum intra-batch coefficient of variation (CV) of 5 positive sera in the repeatability test was 1.08% and the maximum was 5.55%, while the minimum inter-batch CV was 1.64% and the maximum was 5.64%, both less than 10% (Table 8). The results of intra-batch and inter-batch repeatability tests using recombinant protein S1-II as the coating antigen showed that the minimum intra-batch CV of 5 positive sera in the repeatability test was 2.12% and the maximum was 5.27%, while the minimum inter-batch CV was 1.56% and the maximum was 5.38%, both less than 10% (Table 9). The results showed that the experimental repeatability of recombinant protein S1-I as the coating antigen was better.

[0095] Table 8: Intra-batch and inter-batch reproducibility results of recombinant protein S1-I

[0096]

[0097] Table 9: Intra-batch and inter-batch reproducibility results of recombinant protein S1-II

[0098]

[0099] 6) Sensitivity test

[0100] The GI-19 IBV antibody positive serum was diluted from 1:1000 to 1:128000 to determine the positive judgment standard and the antibody titer of the positive serum was tested.

[0101] Recombinant protein S1-I was used as the coating antigen for sensitivity experiments. IBV positive serum was diluted from 1:1000 for indirect ELISA detection. When the primary antibody serum dilution was 1:32000, the OD450 value was still higher than the judgment standard. Recombinant protein S1-II was used as the coating antigen for sensitivity experiments. IBV positive serum was diluted from 1:1000 for indirect ELISA detection. When the primary antibody serum dilution was 1:16000, the OD450 value was still higher than the judgment standard. Recombinant protein S1-I as the coating antigen has higher sensitivity.

[0102] 7) Specificity test

[0103] The established indirect ELISA method was used to detect AIV (H5), AIV (H9), NDV, GAstV, IBV (GVI-1), IBV (H120), and IBV (ahysx-1) positive sera, respectively. At the same time, IBV positive sera were set up as controls with negative sera. Each sample was repeated 3 times to determine whether the method had any cross-reaction.

[0104] The results showed that the recombinant protein S1-I was used as the coating antigen for indirect ELISA detection of AIV (H5), AIV (H9), NDV, GAstV positive serum and SPF serum. Except for IBV positive serum, the S / P values ​​of other sera were all lower than the critical value of 0.097 (Table 10). The recombinant protein S1-I was used as the coating antigen for indirect ELISA detection of AIV (H5), AIV (H9), NDV, GAstV positive serum and SPF serum. Except for IBV positive serum, the S / P values ​​of other sera were all lower than the critical value of 0.136 (Table 11). The recombinant protein S1-I was used as the coating antigen only to produce a good reaction with GI-19 type IBV positive serum, while the P / N value of the detection results for GVI-1 type IBV, H120 type IBV, and ahysx-1 type IBV positive serum was low. This result shows that S1-I has good serotype specificity as a detection antigen, and has little cross-reaction with non-GI-19 type IBV antibodies.

[0105] Table 10: Recombinant protein S1-I specificity test results

[0106]

[0107] Table 11: Results of specificity experiments of recombinant protein S1-II

[0108]

[0109] In summary, the genetically engineered recombinant GI-19 IBV antigen epitope polypeptide S1-I provided by the present invention can specifically bind to GI-19 IBV positive serum, and has a weak cross-reaction with non-GI-19 IBV positive serum, and can be used for the detection and evaluation of GI-19 IB vaccine immune antibodies and clinical GI-19 IBV infection monitoring.

Claims

1. A chicken infectious bronchitis virus antigen epitope fusion peptide, characterized in that: The amino acid sequence of the antigen epitope fusion peptide is SEQ ID NO:

4.

2. The fusion peptide of avian infectious bronchitis virus antigen epitope according to claim 1, characterized in that: The nucleotide sequence encoding the antigen epitope fusion peptide is SEQ ID NO:

3.

3. The fusion peptide of avian infectious bronchitis virus antigen epitope according to claim 1, characterized in that: The nucleotide sequence encoding the antigen epitope fusion peptide is SEQ ID NO:

6.

4. A recombinant expression vector, characterized in that: The recombinant expression vector is inserted with a nucleic acid fragment encoding the antigen epitope fusion peptide according to claim 1.

5. A prokaryotic expression host bacteria, characterized in that: The prokaryotic expression host bacteria carries the recombinant expression vector according to claim 4.

6. Use of the antigen epitope fusion peptide according to claim 1 as a coating antigen in the preparation of an indirect ELISA detection product.

7. An indirect ELISA detection product, characterized in that, The coated antigen in the indirect ELISA detection product is the antigen epitope fusion peptide according to claim 1.

8. The indirect ELISA detection product according to claim 7, characterized in that, The antigen coating concentration is 0.5-1 μg / mL.

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