Bovine influenza A virus H5 subtype cELISA antibody detection method

Abstract

According to the bovine influenza A virus H5 subtype cELISA antibody detection method, on the basis that specific antigen fragments are obtained through screening, the influenza A virus H5 subtype antibody detection method which is convenient, rapid, high in sensitivity and high in specificity is established. The amino acid sequence of the antigen protein is SEQ ID NO: 2. The antigen protein screened by the invention is good in specificity and high in sensitivity, is only specifically combined with corresponding subtype bovine influenza virus positive serum, and does not generate cross reaction with other epidemic disease positive serum. And when the bovine influenza A virus H5 subtype cELISA antibody detection method is used for detecting the AIV antibody, the detection can be conveniently and quickly carried out, and the result judgment is accurate and reliable. The cost is low, batch detection can be realized in one step, the cost is low, and the effect is quick.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology detection products, and particularly relates to a cELISA antibody detection method for bovine influenza virus A H5 subtype. BACKGROUND

[0002] Influenza virus is a virus of Orthomyxoviridae, and can be divided into influenza virus A, B, C and D according to the antigen characteristics of nucleoprotein (NP) and matrix protein (M1). According to the latest literature, the host of influenza virus D is mainly pigs, and no human infection case has been reported. The host of influenza virus C is human and pig, and it mainly infects children under the age of six in the human population, and usually causes fever and mild respiratory tract infection. Influenza virus A and B cause seasonal epidemics every year, and can cause global pandemic in severe cases. After being infected with influenza virus, the symptoms of mild cases are muscle soreness, cough, runny nose, rapid high fever, and most patients also have more serious pneumonia symptoms. Patients with more serious symptoms will have multiple organ failure such as heart and kidney, and eventually die. Influenza virus A and B have a wide range of transmission and high mortality, and seasonal influenza infects 300 to 500 million people worldwide every year, resulting in 290,000 to 650,000 deaths. Influenza virus A has the widest host range and variation degree, and can infect a variety of birds and mammals, and is the main pathogen of seasonal influenza epidemic and occasional pandemic in human population.

[0003] Highly pathogenic avian influenza is a major animal disease that seriously endangers the healthy development of China's poultry industry. It is listed as a statutory report disease by the World Organization for Animal Health (WOAH), and is listed as a class I animal disease in China. At present, the global highly pathogenic avian influenza epidemic situation is severe, and the epidemic situation frequently occurs in Japan, India, South Korea and Vietnam and other neighboring countries, and the risk of spread by migratory birds is relatively large. Highly pathogenic avian influenza shows a point distribution trend in the Asia-Pacific region, and there is a risk of cross-regional transmission. Early warning monitoring, information exchange and sharing should be strengthened to achieve early detection, early disposal and early prevention.

[0004] A type H5N1 subtype avian influenza virus is a highly pathogenic subtype of influenza A virus with hemagglutinin (hemagglutinin) type 5 and neuraminidase (neuraminidase) type 1. In recent years, H5N1 subtype avian influenza virus has spread worldwide and infected humans and various animals. In the influenza surveillance of chickens, various serological antibody detection techniques have been established, and the key methods are: agar diffusion test (AGP), hemagglutination inhibition test (HI) and indirect enzyme-linked immunosorbent assay (ELISA). In March 2024, Texas, USA, reported for the first time that dairy cows were infected with 2.3.4.4b H5N1 highly pathogenic avian influenza, and the virus belongs to B3.13 genotype of 2.3.4.4b lineage. This genotype of virus currently only exists in the United States. As of March 31, 2025, there were 995 dairy farms in 17 states in the United States that were diagnosed with H5N1 outbreaks. Studies have shown that H5N1 can be directly transmitted from wild birds to dairy cows across hosts, and the continued outbreak of H5N1 avian influenza poses an increasing threat to dairy cows. Research has found that H5N1 virus not only causes dairy cows to exhibit obvious clinical symptoms, but also replicates and excretes the virus in cow's milk efficiently. These findings reveal the potential risk of virus transmission in livestock and the possible threat to public health. There have been no reports of domestic cows being infected with H5N1 subtype avian influenza virus, but it is very important to reserve related detection methods. SUMMARY

[0005] The purpose of the present application is to provide a method for detecting antibodies of A type influenza virus H5 subtype in cows. Based on the screening of specific antigen fragments, a convenient, rapid, sensitive and specific antibody detection tool and detection method for detecting A type influenza virus H5 subtype are established.

[0006] The present application first provides an antigen protein for detecting A type influenza virus H5 subtype in cows, wherein the amino acid sequence of the antigen protein is SEQ ID NO: 2.

[0007] The present application further provides a nucleic acid fragment for encoding the above-mentioned protein, and a specific nucleotide sequence thereof is SEQ ID NO: 1.

[0008] The present application further provides a recombinant strain, wherein the recombinant strain comprises a recombinant expression vector into which the above-mentioned nucleic acid fragment is inserted.

[0009] In another aspect, the present application further provides a monoclonal antibody, which is prepared by immunizing mice with the above-mentioned antigen protein.

[0010] The present application further provides a kit for detecting whether a cow is infected with A type influenza virus H5 subtype, which comprises the above-mentioned antigen protein and monoclonal antibody.

[0011] The monoclonal antibody further comprises positive serum and negative reference serum.

[0012] Further, the kit is a cELISA antibody detection kit.

[0013] The application further provides a method for detecting whether a cow is infected with influenza A virus H5 subtype, which uses the cELISA antibody detection kit.

[0014] The antigen protein screened by the application has good specificity and high sensitivity, and is specifically combined with corresponding influenza A virus H5 subtype positive serum, and does not have cross reaction with other disease positive serum. When the cELISA antibody detection method is used to detect AIV antibody, the detection can be conveniently and quickly performed, the result is accurate and reliable, the cost is low, batch detection can be performed, one step is achieved, the cost is low, and the effect is fast. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Figure 1 is an expression and location diagram of the recombinant plasmid pKG-H5, wherein lane M is Proterin Marker, lane 1 is expression of pKG-H5 in BL21, lane 2 is expression of pGEX-H5 in BL21, lane 3 is expression of pKG-H5 in supernatant of the recombinant bacteria, and lane 4 is expression of pKG-H5 in precipitate of the recombinant bacteria;

[0016] Figure 2 Figure 2 is a PCR identification diagram of the recombinant bacteria AIV-H5 hemagglutinin, wherein lane M is Marker 2000, lanes 1-8 are amplification product fragments of the AIV-H5 hemagglutinin gene of the 1st, 3rd, 5th, 7th, 9th, 10th, 11th and 12th generations of the recombinant bacteria, lane 9 is a positive control, and lane 10 is a negative control;

[0017] Figure 3 Figure 3 is a detection diagram of the expression amount of the target protein of the recombinant bacteria of different generations, wherein lane M is Proterin Marker, lane 1 is expression product of the empty vector in BL21, and lanes 2-9 are SDS-PAGE detection of the protein expression of the 1st, 3rd, 5th, 7th, 9th, 10th, 11th and 12th generations of the recombinant bacteria;

[0018] Figure 4 Figure 4 is a detection diagram of the expression amount of the target protein of the recombinant bacteria, wherein lane 1 is pGEX-H5 empty vector control, and lanes 2-9 are western detection of the expression protein of the 1st, 3rd, 5th, 7th, 9th, 10th, 11th and 12th generations of the recombinant bacteria. DETAILED DESCRIPTION

[0019] The core advantage of the competition ELISA method is that it is a standard method for detecting small molecules, haptens or single epitope analytes, and is suitable for small molecule antigens, is relatively resistant to complex sample matrix, and has good sensitivity. When high-affinity and high-specificity antibodies are used, the competition method can detect small molecule analytes at extremely low concentrations (such as pg / mL or even fg / mL levels), has relatively simple operation steps, short time, small reagent consumption, and relatively low cost.

[0020] The present application designs a specific protein fragment according to the hemagglutinin of the H5 subtype virus, and detects the H5 subtype antibody of the A type influenza virus in cattle by using a cELISA method, so as to diagnose the infection of the A type influenza virus H5 subtype in cattle.

[0021] The present application screens the hemagglutinin protein of the H5 subtype, and constructs a recombinant E. coli BL21 / H5 which efficiently and stably expresses the gene. The morphological characteristics and culture characteristics of the strain are consistent with those of the recipient strain (E. coli BL21), and the strain has high safety. After the expressed protein product is purified, a cELISA diagnosis method for detecting the H5 subtype antibody of the A type influenza virus in cattle is established.

[0022] Since the traditional fixed value determination method is easily affected by human factors (such as operator technical errors) and environmental factors (such as environmental temperature), false positive and false negative results are easily generated. In order to avoid the generation of the above factors, the determination standard of the cELISA antibody detection method for the H5 subtype of the A type influenza virus in cattle is established by using a blocking rate method, and the determination formula is as follows: the OD450nm values of each well are read on an enzyme label instrument. The test is established under the condition that the OD450nm values of the negative control wells are all greater than or equal to 0.5, and the PI values of the positive control wells are all greater than 55%. The PI value of the sample is calculated.

[0023] PI = (OD450nm value of negative control - OD450nm value of sample) ÷ OD450nm value of negative control × 100%

[0024] If PI is greater than or equal to 55%, it is determined that the H5 subtype antibody of the A type influenza virus is positive; if PI is less than 55%, it is determined that the H5 subtype antibody of the A type influenza virus is negative.

[0025] Specific detection is performed by using the established competition ELISA test method, and the results show that only the H5 subtype AIV reacts, and other subtypes of AIV and NDV do not react, and the specificity is good. Detection of H1, H3, H4, H5, H6, H7, H8, H9, H10 subtype and NDV positive serum shows that the PI values of H1, H3, H4, H6, H7, H8, H9, H10 subtype and NDV serum are all less than the negative critical value (55%), and are all determined to be negative. Only the PI value of the H5 subtype is greater than the positive critical value (55%), and is determined to be positive.

[0026] The broad-spectrum detection was performed by using the established competitive ELISA test method, and it was shown that the detection method had good reaction to different H5 influenza virus epidemic branches and had broad-spectrum reaction reactivity to H5 subtype AIV. The H5 subtype Re-4, Re-5, Re-6, Re-7, Re-8, Re-10, Re-11, Re-12 epidemic branch positive serum was detected, and the SPF chicken serum was used as a control. The results showed that the PI value of the detection method for H5 subtype Re-4, Re-5, Re-6, Re-7, Re-8, Re-10, Re-11, Re-12 immune serum was greater than the positive critical value (55%), and was determined to be positive.

[0027] The application will be described in detail below in combination with examples and drawings.

[0028] Example 1: Construction and biological characteristics of recombinant E. coli BL21 / H5 expressing H5 subtype protein of influenza A virus

[0029] 1. RT-PCR amplification of AIV-H5 hemagglutinin protein gene

[0030] According to the nucleotide sequence of the AIV-H5 hemagglutinin protein gene, a pair of specific primers for amplifying the AIV-H5 hemagglutinin protein gene were designed, and EcoR I and Xho I enzyme digestion sites were introduced into the upstream and downstream primers, respectively. The reaction conditions were as follows: after pre-denaturation at 94℃ for 5 minutes, PCR cycles were performed, with denaturation at 94℃ for 1 minute, annealing at 55℃ for 1 minute, extension at 72℃ for 1 minute, for a total of 35 cycles, and finally extension at 72℃ for 10 minutes.

[0031] Upstream primer F: 5'-atgatatcatggaaaacatagtacttctt-3'

[0032] Downstream primer R: 5'-atctcgagctaaatgcaaattctg-3'

[0033] The nucleotide sequence of the AIV-H5 hemagglutinin protein amplified fragment is as follows:

[0034] atggaaaacatagtacttcttcttgcaataattagccttgttaaaagtgatcagatttgcattggttaccatgcaaacaat

[0035] tcgacagagcaggttgacacgataatggaaaagaacgtcaccgttacacatgcccaagacatactggaaaaagcacaca

[0036] acgggaagctctgtgatctaaatggggtgaagcctctgattttaaaggattgtagtgtagctggatggcttctcggaaaccc

[0037] aatgtgcgacgaattcatcagagtgccggaatggtcctacatagtggagcgggccaatccagctaatgacctctgttaccc

[0038] agggagcctcaatgactacgaagaactgaaacacctgttgagcagaataaatcactttgagaagattctgatcatccccaa

[0039] gagttcctggccaaatcatgaaacatcattaggggtgagcgcagcttgtccataccagggagcgccctcctttttcagaaat

[0040] gtggtgtggcttatcaaaaagaacgatgcatacccaacaataaagataagctacaataataccaatcgggaagatctcttga

[0041] tactgtgggggattcatcattccaacaatgcagaagagcagacaaatctctataaaaatccaaccacctacatttcagttgga

[0042] acatcaactttaaaccagaggttggtaccaaaaatagctactaggtcccaagtaaacgggcaacgtggaagaatggacttc

[0043] ttctggacaattttaaaaccagatgatgccatccatttcgagagcaatggaaatttcattgctccagaatatgcatacaaaattg

[0044] tcaagaaaggggactcaacaattatgaaaagtggagtggaatatggccactgcaacaccaaatgtcaaaccccagtagga

[0045] GCGATGATTTCTAGTAAGCCATTCCACAACATACTACCTCTCACCATTGGGGAAACCCCTAAATATGTGAAATCAAACAAGTT

[0046] GGTCCTTGC GACTGGGCTC AGAAATAATCCTCTAAGAGAAAGGAGAAGAAAAAGAGGCCTGT TTGGGGCGATAGCAGGG

[0047] TTTATAGAGG GAGGATGGCA AGGAATGGTT GATGGTTGGT ATGGGTACCATCATAGCAATGAGCAGGGGAGTGGGTACGC

[0048] TGCAGACAAA GAATCCACCC AAAAGGCAAT AGATGGAGTT ACCAATAAGGTC AACTCAATCA TTGACAAAAT GAACACTCA

[0049] ATTTGAGGC AGTTGGAAGG GAGTTTAATA ACTTAGAAAG GAGGATAGAG AATTTGAACA AGAAAATGGA AGACGGATTCCT

[0050] AGATGTCTGGACCTATAATGCTGAAC TTCTAGTTCT CATGGAAAAC GAGAGGACTC TAGATTTCAT GATTCAATGT CAAGAA

[0051] TCTTTACGAC AAAGTCAGAC TACAGCTTAG GGATAATGCA AAGGAGCTGG GTAATGGCTG TTTCGAATTC TATCACAAAT GCG

[0052] ATGATGAAAT ATGGAAAGTG TGAGAAATGG GACGTATGAC TACCCTCAGT ATTCAGAAGA AGCAAGATTA AAAAGAGAAG

[0053] AAATAAGCGGAGTGAAATTAGAATCAATAGGAAC TTACCAGAT ACTGTCAATT TATTCAACAG CGGCAGTTCC CTA GC ACT

[0054] GGCAATCATGATGGCTGCTCTATCTTTATGGATGTGCTCCAATGGGTCGTTCGTGCAGAATTTCATTCTAG (SEQ ID NO: 1);

[0055] The amino acid sequence of the polypeptide encoding AIV-H5 hemagglutinin protein is as follows:

[0056] MENIVLLLAIISLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKAHNGKLCDLNG

[0057] VKPLILKDCSVAGWLLGNPMCDEFIRVPEWSYIVERANPANDLCYPGSLNDYEELKHLLSRI

[0058] NHFEKILIIPKSSWPNHETSLGVSAACPYQGAPSFFRNVVWLIKKNDAYPTIKISYNNTNRED

[0059] LLILWGIHHSNNAEEQTNLYKNPTTYISVGTSTLNQRLVPKIATRSQVNGQRGRMDFFWTIL

[0060] KPDDAIHFESNGNFIAPEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSMPFHNIH

[0061] PLTIGECPKYVKSNKLVLATGLRNNPLRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHH

[0062] SNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDG

[0063] FLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDD

[0064] ECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTAASSLALAIMMAGLSL

[0065] WMCSNGSLQCRICI (SEQ ID NO: 2).

[0066] 2. Construction and identification of recombinant plasmid pKG-H5

[0067] The amplified product of AIV-H5 hemagglutinin protein gene was digested with EcoRI and Xhol, and the fragment of about 1704 bp was recovered and ligated with the prokaryotic expression vector pGEX-KG digested with EcoRI and Xhol. The ligation product was transformed into E. coli DH5α, and the positive recombinant plasmid was identified by enzyme digestion and named pKG-H5.

[0068] 3. Induced expression of recombinant plasmid pKG-H5

[0069] The recombinant plasmid pKG-H5 was transformed into E. coli BL21, and IPTG was added for induced expression. The samples were taken every 1 hour from 3 hours after induction, and the expression was detected by SDS-PAGE electrophoresis. The results showed that no specific expression protein was detected in the uninduced sample, but specific expression bands were detected in the samples induced for different times, and the protein expression was the highest at 3 hours after induction. Therefore, 3 hours was determined as the optimal induction time.

[0070] 4. Localization of expression of recombinant plasmid

[0071] The induced bacterial solution was centrifuged at 8000 r / min for 10 minutes, and the bacterial body was collected and suspended in 20 ml buffer A (50 mmol / l Tris-HCl pH 8.0, 0.5 mmol / L EDTA pH 8.0, 50 mmol / L NaCl, 0.5 mmol / L dithiothreitol (DTT), 5% glycerol). The bacterial body sample was placed on ice and was fully broken by ultrasonic waves until the solution became clear and no longer viscous. The broken solution was centrifuged at 10000 r / min for 15 minutes, and the supernatant and the precipitate were collected. SDS-PAGE electrophoresis detection showed that a specific protein band appeared at about 42 KD in the precipitate of the recombinant bacteria, which was consistent with the expected size (Fig. 2), and no specific band was observed in the control, and the protein was mainly expressed in the inclusion body. Figure 1

[0072] 5. Extraction of inclusion body and detection of expression product

[0073] ​Western-blot detection with H5 subtype monoclonal antibody as the primary antibody, the results in 42KD appeared a specific band, indicating that AIV-H5 hemagglutinin protein gene in E. coli was highly expressed, and mainly in the form of inclusion bodies, and has good biological activity. Extract the inclusion bodies and purify, using polyethylene glycol 20000 concentrated protein to the original LB liquid medium volume of sixty-sixth, ultraviolet spectrophotometer determination of protein at 280nm and 260nm wavelength (OD), according to formula 1.45xOD 280nm -0.74xOD 260nm The protein concentration was calculated as 2.82mg / ml.

[0074] 6、Recombinant bacteria BL21 / H5 test

[0075] According to the current "Chinese Veterinary Pharmacopoeia" appendix, the recombinant bacteria were detected, and the results confirmed that the recombinant bacteria did not contain other bacteria, and the test results were pure. The recombinant bacteria were cultured in ordinary medium for 12-16 hours, and grew well, with smooth grayish white colonies and a diameter of 2.5mm.

[0076] The above results show that the AIV-H5 hemagglutinin protein gene with the sequence of SEQ ID NO: 1 is inserted into the prokaryotic expression vector pGEX-KG to obtain the prokaryotic expression plasmid pKG-H5 expressing the AIV-H5 hemagglutinin protein with the amino acid sequence of SEQ ID NO: 2. The plasmid is transformed into E. coli BL21 to obtain the recombinant strain BL21 / H5 expressing AIV-H5 hemagglutinin protein.

[0077] Example 2: Establishment of seed batch of engineering bacteria for bovine influenza virus type A H5 subtype cELISA antibody detection method

[0078] 1、Bacteria AIV-H5 hemagglutinin gene PCR identification

[0079] The bacteria were subcultured in LB solid medium, and the AIV-H5 hemagglutinin gene was amplified with each generation of bacteria as the template. The reaction conditions were as follows: 94℃ pre-denaturation for 5 minutes, then PCR cycle, cycle parameters were 94℃ denaturation for 1 minute, 55℃ annealing for 1 minute, 72℃ extension for 1 minute, a total of 35 cycles, and finally 72℃ extension for 10 minutes.

[0080] The recombinant bacteria BL21 / H5 subcultured in LB solid medium were subcultured for 12 generations, and the AIV-H5 hemagglutinin gene was amplified with one single colony of the 1st, 3rd, 5th, 7th, 9th, 10th, 11th, 12th generation as the template. The results showed that the 12 generations of bacteria could stably amplify the AIV-H5 hemagglutinin gene. Figure 2

[0081] ​2. Detection of the expression amount and titer of the target protein of different generations of the recombinant bacteria

[0082] The inclusion bodies extracted from the 12th generation of the recombinant bacteria after induction were subjected to SDS-PAGE detection, and a specific protein band with a size of about 42 kDa appeared Figure 3 . Western blot detection confirmed that the expression products of the first 12 generations of bacteria had biological activity, and the titer of the expression protein was 1:640 Figure 4 . The amount of expression protein and the titer of protein both decreased from the 12th generation (Table 1).

[0083] Table 1: Detection table of genetic stability of the recombinant bacteria

[0084]

[0085] Note: "S" indicates that the detection result is positive, indicating that the morphological and biochemical characteristics of the bacteria meet the standard of E. coli or the strain is pure;

[0086] " +" indicates that the detection result is positive, indicating that the p54 gene can be amplified or the p54 protein can be detected;

[0087] "-" indicates that the detection result is negative, indicating that the strain does not meet the standard or the p54 gene cannot be amplified or the p54 protein cannot be detected;

[0088] Protein titer: the maximum dilution of the positive control serum protein can be detected.

[0089] 3. Establishment and testing of seed batches

[0090] 3.1 The first generation of propagated seeds was inoculated with a suitable amount of preserved bacteria using a loop, inoculated on LB agar plates containing 100 μg / ml ampicillin, and incubated at 37°C for 12 hours. At least 5 typical colonies were selected and streaked onto LB agar plates containing 100 μg / ml ampicillin (one plate for each single colony), and incubated at 37°C and 200 r / min for 12 hours. The testing was qualified according to the standard of 3.1. The qualified colonies were placed in a refrigerator at 2-8°C for storage as the first generation of seeds, which were still stable and alive within 15 days.

[0091] 3.2 The second generation of propagated seeds was inoculated with the first generation of seeds (single colony) in 2 ml of LB liquid medium containing 100 μg / ml ampicillin, incubated at 37°C for 8 hours, and then stored in a refrigerator at 2-8°C as the second generation of seeds (production seeds). The second generation of propagated seeds (production seeds) were inoculated and induced the next day, and all could express recombinant AIV-H5 hemagglutinin protein with a size of about 42 kDa.

[0092] 3.3 Results of the shelf life test of the strain The basic strains stored at -70°C after freeze-drying were revived every 6 months within 66 months. The AIV-H5 hemagglutinin gene was amplified by PCR, and a fragment of approximately 1704 bp was obtained. At the same time, the expression of each revived strain was induced, and SDS-PAGE and Western-blot detection confirmed that a 42 kDa expression product was obtained, which had biological activity. This shows that the freeze-dried strain is stable after 24 months of storage at -70°C. In order to ensure the preservation effect of the strain, its shelf life is tentatively set at 18 months (Table 2).

[0093] Table 2: Stability test results of seed bacteria during storage period

[0094]

[0095]

[0096] Note: "S" means the test result is positive, indicating that the strain is pure;

[0097] “+” indicates that the AIV-H5 hemagglutinin gene fragment was amplified or there was a specific expression band of the AIV-H5 hemagglutinin protein.

[0098] Genetic stability testing of recombinant BL21 / H5 strains at different passages confirmed genetic stability up to the sixth passage on LB solid medium. PCR amplification of the AIV-H5 hemagglutinin gene was consistently detected in all 12 passages, and all six passages expressed the AIV-H5 hemagglutinin protein encoded by the gene at a titer of 1:640. To ensure sufficient protein for antigen production, the base seed strain was maintained at five passages, as was the production seed strain. The base strain, freeze-dried and stored at -70°C, was revived every three to six months for 24 months, and active expression product was detected at levels exceeding 2.4 mg / ml, demonstrating that the freeze-dried strain can be stored at -70°C for at least 18 months.

[0099] Example 3: Preparation and Standardization of Positive Reference Serum, Negative Reference Serum and Monoclonal Antibodies

[0100] Select 1-2 years old healthy cows, with hemagglutination inhibition test for antibody is negative, inactivated avian influenza H5N1 subtype virus by neck muscle injection, a total of 2 times, interval 14 days, 14 days after the second vaccination every other week through the jugular vein blood separation serum, when the avian influenza virus H5 subtype fluorescence RT-PCR detection method for antigen positive, through the carotid artery aseptic blood, centrifugal separation of serum, 56℃ inactivation 30 minutes, add the final concentration of 0.04% thiomersal sodium preservative, 0.45 μm filter sterilization. -70℃ preservation, the effective period is 24 months. The prepared positive serum with protective agent dilution 10 times, sterile packaging, 0.5ml per tube, namely the method of positive control serum.

[0101] Select 1-2 years old healthy cows, with avian influenza virus H5 subtype fluorescence RT-PCR detection method for antigen negative. Jugular vein aseptic blood, separation of serum, 56℃ inactivation 30 minutes, add 0.04% thiomersal sodium, 0.45 μm filter sterilization. -70℃ preservation, the effective period is 24 months. The negative serum with protective agent dilution 10 times, sterile packaging, 0.5ml per tube, namely the negative control serum.

[0102] Example 4: preparation of monoclonal antibody

[0103] 1. Establishment of anti-H5 AIV HA McAb hybridoma cell strain

[0104] 12 eight-week-old female Balb / C mice were divided into 4 groups, each group of 4 mice. Then the purified H5 subtype AIV protein with amino acid sequence of SEQ ID NO: 2 was mixed with an equal amount of Freund's complete adjuvant for the first immunization, 0.5ml per mouse, 14 days later, the antigen emulsified with Freund's incomplete adjuvant for the second immunization, the same as before. 7 days after the third immunization, blood was taken when the antibody titer reached more than 25, 3-4 days later, the spleen was taken for cell fusion by intraperitoneal injection of antigen without adjuvant 0.5ml.

[0105] 2. Hybridoma cell fusion

[0106] Mix the spleen cells and SP2 / 0 cells in a 50 mL centrifuge tube at the optimal ratio of 7:1, centrifuge at 1000 rpm for 10 min, discard the supernatant, and note that the residual liquid should be completely absorbed to avoid affecting cell fusion; gently rub the centrifuge tube bottom with the palm, mix evenly, and make sure that the cells are not stratified; place the centrifuge tube in a 37°C water bath; slowly add 1 mL of preheated PEG1500 within 1 min, and gently shake to ensure that the cells are fully fused; then, add 30 mL of preheated DMEM medium dropwise within 1 min to terminate the fusion reaction, and stand for 10 min; then, centrifuge at 1000 rpm for 10 min, discard the supernatant, add an appropriate amount of HAT medium, resuspend, and then gently place the feeder cell plate; then, place it in a 37°C cell culture incubator containing 5% CO2. After fusion, observe the cell growth state every day, and avoid unnecessary movement. Four days after fusion, aspirate half of the supernatant in the 96-well cell culture plate, and add HAT medium; seven days after fusion, aspirate all the supernatant, and add HT medium; when the supernatant turns yellow or white cell clumps appear at the bottom, perform antibody detection.

[0107] 3. Preparation of monoclonal antibody ascites

[0108] Take 5 Balb / C female mice, and inject 0.5 mL of sterile liquid paraffin into the abdominal cavity of each mouse. After 7 days, collect the hybridoma cells that grow well and are in the logarithmic growth phase, resuspend them in 37°C preheated normal saline, and count the cells. Inject 1 x 10 6 hybridoma cells into the abdominal cavity of each mouse at 0.5 mL per mouse. Observe the growth state of the mice every day, and after 7-10 days, the mice's abdomens will swell, and will have a wavy feeling when touched. Use a 16-gauge needle soaked in 75% alcohol to puncture the mouse's abdomen to collect the ascites, and note that the needle should be inserted from both sides of the abdomen to avoid internal organs. The ascites can be collected multiple times a day. Centrifuge the collected ascites at 12000 rpm and 4°C for 10 min to remove grease and other impurities, aspirate the supernatant, and then divide it into finger-shaped tubes. Then, use the protein A (SPA) method to purify IgG from the ascites and the positive hybridoma cell culture supernatant, and store them at -20°C for later use.

[0109] Example 5: Specificity and broad-spectrum detection of the cELISA antibody detection method

[0110] Use the established competitive ELISA test method to detect H1, H3, H4, H5, H6, H7, H8, H9, H10 subtypes, and NDV positive serum. The results show that the PI values of H1, H3, H4, H6, H7, H8, H9, H10 subtypes, and NDV serum are all less than the negative critical value (55%), and are all determined to be negative. Only the PI value of H5 subtype is greater than the positive critical value (55%), and is determined to be positive (Table 3).

[0111] Table 3: Results of specific detection by competition ELISA

[0112]

[0113] The established competition ELISA test method was used to detect H5 subtype Re-4, Re-5, Re-6, Re-7, Re-8, Re-10, Re-11, Re-12 positive serum, and SPF chicken serum was used as a control. The results showed that the PI value of the detection method for H5 subtype Re-4, Re-5, Re-6, Re-7, Re-8, Re-10, Re-11, Re-12 immune serum was greater than the positive critical value (55%), and was determined to be positive (Table 4).

[0114] Table 4: Results of broad-spectrum detection by competition ELISA

[0115]

[0116] Example 6: Establishment of ELISA antibody detection method for bovine influenza virus H5 subtype

[0117] The method is composed of antigen-coated plates (96 wells / plate x 2 plates), positive control serum (0.5 ml / tube x 1 tube), negative control serum (0.5 ml / tube x 1 tube), sample diluent (24 ml / bottle x 1 bottle), 20-fold concentrated washing solution (30 ml / bottle x 1 bottle), monoclonal antibody-biotin (12 ml / bottle x 1 bottle), avidin-enzyme label (24 ml / bottle x 1 bottle), substrate solution (24 ml / bottle x 1 bottle), stop solution (24 ml / bottle x 1 bottle), and 1 copy of instructions.

[0118] 1. Preparation of ELISA related reagents

[0119] (1) 20-fold concentrated washing solution 170 g of sodium chloride, 10 ml of Tween-20, and 0.2 g of sodium thiomersalate were dissolved in water for injection and made up to 1000 ml, and divided into 30 ml / bottle, and stored at 2-8°C for standby use.

[0120] (2) Monoclonal antibody-biotin The biotin-labeled H5 subtype monoclonal antibody was diluted 1:5000 times with antibody protectant, filtered to remove bacteria, sterilely divided, 12 ml / bottle, and stored at 2-8°C for standby use.

[0121] (3) Avidin-enzyme label The avidin-labeled horseradish peroxidase was diluted 1:5000 times with protectant, filtered to remove bacteria, sterilely divided, 24 ml / bottle, and stored at 2-8°C for standby use.

[0122] (4)Blocking solution: Bovine serum albumin (BSA) 5g, sodium chloride (NaCl) 8.0g, sodium phosphate dibasic (Na2HPO4-12H2O) 2.9g, potassium phosphate monobasic (KH2PO4) 0.2g, potassium chloride (KCl) 0.2g, dissolved in water for injection and diluted to 1000ml, and stored at 2-8°C for standby.

[0123] (5)Substrate solution: Tetramethyl benzidine was divided into 24ml per bottle.

[0124] (6)Stopping solution: 9ml hydrochloric acid (HCl) was added to 900ml water for injection, and diluted to 1000ml, and divided into 24ml per bottle.

[0125] (7)Sample dilution solution: Bovine serum albumin (BSA) 5g, Tween-20 0.5ml, sodium chloride (NaCl) 8.0g, sodium phosphate dibasic (Na2HPO4-12H2O) 2.9g, potassium phosphate monobasic (KH2PO4) 0.2g, potassium chloride (KCl) 0.2g, sodium thiomersal 0.2g, sterile skim milk powder 0.5g, dissolved in water for injection and diluted to 1000ml, and divided into 24ml per bottle.

[0126] (8)Protective agent: BSA 0.5g, sucrose 2g, dissolved in water for injection and diluted to 100ml, and stored at 2-8°C for standby.

[0127] 2. Preparation and packaging of antigen-coated plates

[0128] a Dilute the antigen to the working concentration with the coating solution, coat the enzyme-labeled plate at 100μl per well, and store at 2-8°C for 15 hours.

[0129] b Discard the solution in the well, add 120μl per well of blocking solution, and incubate at 37°C for 2 hours.

[0130] c Dry and dry at 37°C for 2 hours, and seal and package in an aluminum foil bag.

[0131] 3. Preparation, testing and packaging of negative control serum

[0132] a The manufacturing animal is selected from 1-2 year old healthy cows, the antibody is negative by hemagglutination inhibition test, and the antigen is negative by avian influenza virus H5 subtype fluorescent RT-PCR detection method.

[0133] b Serum collection and treatment: aseptically collect blood from the jugular vein, separate the serum, inactivate at 56°C for 30 minutes, add 0.04% sodium thiomersal, filter with a 0.45μm filter to remove bacteria, store at -70°C, and the effective period is 24 months. Dilute the negative serum 10 times with the protective agent, sterilely package, and 0.5ml per tube, which is the negative control serum.

[0134] c Sterile sub-packaging, 0.5 ml / tube.

[0135] 4. Preparation, testing and sub-packaging of positive control serum

[0136] a. The manufacturing animals are selected from healthy cows aged 1-2 years, with negative antibody by hemagglutination inhibition test and negative antigen by avian influenza virus H5 subtype fluorescent RT-PCR detection method.

[0137] b. The immunogen is inactivated avian influenza H5N1 subtype virus prepared by China Animal Health and Epidemiology Center.

[0138] c. The immunization method is intramuscular injection of vaccine through the neck, with a total of 2 inoculations, 14 days apart, and blood collection through the jugular vein every other week starting 14 days after the second immunization to separate serum.

[0139] d. Serum collection and processing When the test serum is positive, sterile blood is collected through the carotid artery, the serum is separated by centrifugation, inactivated at 56°C for 30 minutes, preserved with 0.04% sodium thiomersal at a final concentration, and filtered through a 0.45 μm filter to remove bacteria. Store at -70°C, with a validity period of 24 months. Dilute the prepared positive serum 10 times with a protective agent, and sterilely sub-packaging, 0.5 ml / tube, which is the positive control serum of the method.

[0140] e. Sterile sub-packaging, 0.5 ml / tube.

[0141] 5. Preparation and sub-packaging of 20-fold concentrated washing solution Dissolve sodium chloride 170 g, 10 ml Tween-20 (Tween-20), sodium thiomersal 0.2 g in water for injection and make up to 1000 ml, and sub-packaging into 48 ml / bottle.

[0142] 6. Preparation and sub-packaging of sample diluent Dissolve bovine serum albumin (BSA) 5 g, Tween-20 (Tween-20) 0.5 ml, sodium chloride 8.0 g, disodium phosphate dodecahydrate (Na2HPO4·12H2O) 2.9 g, potassium dihydrogen phosphate (KH2PO4) 0.2 g, potassium chloride (KCl) 0.2 g, sodium thiomersal 0.2 g, and sterile skim milk powder 0.5 g in water for injection and make up to 1000 ml. Sub-packaging into 24 ml / bottle.

[0143] 7. Preparation and testing of H5 subtype monoclonal antibody-biotin (mAb-biotin)

[0144] a. Preparation method of H5 subtype monoclonal antibody-biotin The H5 subtype monoclonal antibody-biotin used is produced by Pujian Bio.

[0145] b. Sterile sub-packaging, 12 ml / tube.

[0146] c Titer test H5 subtype monoclonal antibody-biotin conjugation method The rest of the qualified reagents were repeated 8 wells to detect the positive reference serum, and the PI value should be 80% ± 10%. The negative reference serum was detected by repeating 8 wells, and the OD450nm value should be > 1.5.

[0147] 8. Preparation and test of avidin horseradish peroxidase

[0148] a Preparation of avidin horseradish peroxidase (avidin-enzyme label) The goat anti-pig IgG-HRP enzyme-labeled antibody used in the trial method was a product of Southern biotechnology associates (SBA) company. After dilution with a protective agent at 1:5000 to the working concentration, it was filtered with a 0.45 μm filter to remove bacteria, sterilely packaged, and 24 ml / bottle.

[0149] b Titer test of avidin horseradish peroxidase conjugation method The rest of the qualified reagents were repeated 8 wells to detect the positive reference serum, and the PI value should be 80% ± 10%. The negative reference serum was detected by repeating 8 wells, and the OD450nm value should be > 1.5.

[0150] c Preparation of substrate solution and stop solution

[0151] d The substrate solution was sterilely packaged into 24 ml / bottle.

[0152] e Preparation of stop solution Hydrochloric acid (HCl) 9 ml, add water for injection to 1000 ml. Package into 24 ml / bottle.

[0153] Example 7: Detection steps of bovine influenza virus type A H5 subtype ELISA antibody detection method

[0154] 1 Step (1) Method After warming, take the antigen-coated plate (according to the amount of sample, it can be disassembled and used in batches)

[0155] (2) Take 50 μl of the diluted sample to be tested, negative control serum and positive control serum, and add them to the antigen-coated plate. The test serum is set in one hole, and the negative and positive controls are each set in two holes. Add 50 μl of monoclonal antibody-biotin to each hole, gently shake the sample in the hole, and incubate at 37°C for 60 minutes.

[0156] (3) Discard the solution in the hole of the plate, wash each hole with 300 μl of washing solution for 5 times, and dry the plate hole on the blotting paper after washing.

[0157] (4) Add 100 μl of avidin-enzyme label to each hole, gently shake the sample in the hole, and incubate at 37°C for 30 minutes.

[0158] (5) Discard the solution in the hole of the plate, wash for 5 times, and the method is the same as (3).

[0159] (6) Add 100 μl of substrate solution to each reaction well, and develop color at room temperature (20-25°C) for 15 minutes in the dark.

[0160] (7) Add 100 μl of stop solution to each reaction well, and measure the results within 10 minutes.

[0161] 2 Judgment

[0162] Read the OD450nm value of each well on the microplate reader. The conditions for the test to be valid are that the OD450nm value of the negative control well should be ≥0.5, and the PI value of the positive control well should be greater than 55%. Calculate the PI value of the sample.

[0163] PI = (OD450nm value of the negative control - OD450nm value of the sample) ÷ OD450nm value of the negative control × 100% If PI ≥ 55%, it is judged to be positive for the antibody of the H5 subtype of influenza virus A; if PI < 55%, it is judged to be negative for the antibody of the H5 subtype of influenza virus A.

[0164] Randomly select 22 serum samples of cows immunized with the inactivated vaccine of the H5 subtype and 2 negative serum samples to detect the accuracy of the method, and the results are shown in Tables 5 and 6.

[0165] Table 5: ELISA detection results of the method of the present application on influenza (IP value)

[0166] Numbering 1 2 3 A 79％ 84％ 82％ B 82％ 85％ 82％ C 84％ 81％ 76％ D 83％ 82％ 84％ E 75％ 79％ 82％ F 80％ 40％ 89％ G 89％ 89％ 22％ H 82％ 80％ 19％

[0167] Table 6: ELISA detection results of the method of the present application on influenza (negative / positive)

[0168]

[0169]

[0170] The results show that the coincidence rate of the method of the present application for detecting the antibody of the H5 subtype of avian influenza virus in the serum of a cow is 95.8%, and the method can efficiently detect whether a cow is infected with the H5 subtype of avian influenza virus.

Claims

1. An antigen protein for detecting bovine influenza A virus H5 subtype, characterized in that: The amino acid sequence of the antigen protein is SEQ ID NO:

2.

2. A nucleic acid fragment, characterized in that The nucleic acid fragment is used to encode the antigen protein according to claim 1.

3. The nucleic acid fragment according to claim 2, wherein The nucleotide sequence of the nucleic acid fragment is SEQ ID NO:

1.

4. A recombinant expression vector, characterized in that: The nucleic acid fragment according to claim 2 is inserted into the recombinant expression vector.

5. A recombinant strain, characterized in that The recombinant strain contains the recombinant expression vector according to claim 4.

6. A monoclonal antibody, characterized in that The monoclonal antibody is prepared by immunizing mice with the antigen protein according to claim 1.

7. A kit for detecting whether cattle are infected with influenza A virus H5 subtype, characterized in that: The kit comprises the antigen protein according to claim 1 and the monoclonal antibody according to claim 6.

8. The kit according to claim 7, wherein The kit is a cELISA antibody detection kit.

9. The kit according to claim 8, wherein The kit also contains positive serum and negative reference serum.

10. A method for detecting whether cattle are infected with influenza A virus H5 subtype, characterized in that: The method is to use the kit according to claim 8 for detection.

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