Recombinant protein for normalized monitoring of H7N9 subtype avian influenza virus in poultry farm and preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a recombinant protein for normalization monitoring of H7N9 subtype avian influenza virus in poultry farms and a preparation method and application of the recombinant protein. DIVA detection polypeptide and Escherichia coli thioredoxin (TrxA) are fused, and the recombinant fusion protein is expressed in a prokaryotic expression system. In addition, the purified polypeptide fusion protein is used as an antigen to establish an ELISA method. The method can only react with the serum of the chicken infected by the H7N9 subtype avian influenza virus. The cost for expressing and purifying recombinant protein in escherichia coli is obviously lower than that of chemically synthesized polypeptide, and the method is suitable for carrying out normalized epidemic disease monitoring in poultry farms. The recombinant fusion protein and the detection method thereof provided by the invention provide cheap and efficient diagnostic antigen and technology for carrying out normalized H7N9 subtype AIV detection in poultry farms.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a recombinant protein for normalized monitoring of H7N9 subtype avian influenza virus in poultry farms, and a preparation method and application thereof. Background Art

[0002] H7N9 subtype avian influenza virus (AIV) is a zoonosis. For local epidemic diseases, regular monitoring is particularly important for grasping the epidemic situation, changes and trends. At present, grassroots epidemic control units, scientific research units or poultry farms mainly use three methods to monitor clinical avian influenza viruses. The first is the virus separation method. This method is the gold standard for disease diagnosis, but the biggest drawback is that the treatment of H7N9 subtype AIV requires biosafety level 3 experimental conditions, which cannot be operated by grassroots testing units and poultry farms. The second is molecular biology methods, such as fluorescent quantitative PCR, to detect viral nucleic acids. This method has high sensitivity and operability, but it also requires high biosafety conditions for the treatment of virus samples, and requires expensive instruments and professionals. The third is serological methods. This method does not require the separation and operation of live viruses, and has strong operability and specificity. It can be integrated with the daily antibody monitoring project of poultry farms, without additional sampling and testing, and can achieve regular monitoring of epidemics. However, conventional serological methods, such as hemagglutination inhibition test and agar diffusion test, cannot diagnose virus-infected animals. Therefore, it is necessary to develop a serological method that can specifically identify H7N9 subtype AIV-infected animals.

[0003] In addition, the cost of diagnostic methods used for routine disease monitoring in poultry farms must be low. On the one hand, the cost of poultry industry inputs must be kept low due to the low value of poultry itself. On the other hand, the high frequency and wide coverage of routine disease monitoring require low-cost related detection methods.

[0004] The prior art discloses a polypeptide that can distinguish H7N9 virus infection from vaccine-immunized animals (DIVA), and establishes a polypeptide-based virus infection detection method. This method can only react with virus-infected chicken serum, but not with vaccine-immunized chicken serum, and can specifically identify virus-infected animals from immune chickens. However, the disclosed technology uses chemically synthesized polypeptides as detection antigens, and the cost of polypeptides is relatively high. Therefore, the cost of the H7N9 virus detection method using chemically synthesized polypeptides as antigens is high, and it is not suitable for the routine monitoring of H7N9 subtype AIV in poultry farms. In addition, the sensitivity of the prior art in detecting H7N9 virus-infected animals is not high. After the serum is diluted 1:200 times, the OD450 ≥ 0.5 with the polypeptide is the critical value for determining virus infection. However, in poultry immunized with vaccines, the virus is often infected at a low level, and the induced antibodies are low, and the critical value of OD450 ≥ 0.5 may cause missed detection. The preparation cycle of the polypeptide antigen required by the prior art is relatively long (generally 3-4 weeks), which reduces the timeliness of disease monitoring. Summary of the invention

[0005] In view of the main problems existing in the existing H7N9 subtype AIV infection detection methods, the present invention fuses the DIVA detection polypeptide with Escherichia coli thioredoxin (TrxA) and expresses the recombinant fusion protein in a prokaryotic expression system. In addition, an ELISA method is established using the purified polypeptide fusion protein as an antigen. This method can only react with the serum of chickens infected with the H7N9 subtype avian influenza virus. The cost of expressing and purifying the recombinant protein in Escherichia coli is significantly lower than that of chemically synthesized polypeptides. The recombinant fusion protein and its detection method provided by the present invention provide a cheap, efficient and sensitive diagnostic antigen and technology for poultry farms to carry out normalized H7N9 subtype AIV detection.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A recombinant protein for routine monitoring of H7N9 subtype avian influenza virus in poultry farms, wherein the amino acid sequence of the recombinant protein is shown in SEQ No. 1, the recombinant protein comprises an H7N9 HA protein polypeptide, an Escherichia coli TrxA protein and a His tag, the amino acid sequence of the H7N9 HA protein polypeptide is shown in SEQ No. 2; the amino acid sequence of the Escherichia coli TrxA protein is shown in SEQ No. 3.

[0008] SEQ No.1:

[0009] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGK

[0010] LTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDA

[0011] NLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFKNVPEIPKGRGLEHHHHHH*;

[0012] SEQ No.2: KNVPEIPKGRG;

[0013] SEQ No.3:

[0014] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGK

[0015] LTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDA NLA;

[0016] The gene sequence encoding the above recombinant protein is shown in SEQ No.4.

[0017] SEQ NO.4 (underlined bases represent TrxA gene sequence; capital letter bases represent HA polypeptide sequence; italic bases represent His tag sequence)

[0018] atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggc gatcctcgtcgatt

[0019] tctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatat cagggcaaactg

[0020] accgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatccc gactctgctgctg

[0021] ttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcct cgacgctaacct

[0022] ggcc ggttctggttctggccatatgcaccatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaa

[0023] ccgctgctgctaaattcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctg

[0024] atatcggatccgAATTCAAGAATGTTCCTGAGATTCCAAAGGGAAGAGGCCtcgagcaccaccaccaccaccactga.

[0025] Furthermore, the gene encoding the H7N9 HA protein polypeptide was cloned into the prokaryotic expression vector pET32a through the restriction sites EcoRI and XhoI.

[0026] Furthermore, an EcoRI site is added to the 5' end of the forward sequence corresponding to the H7N9 HA protein polypeptide, and an XhoI site is added to the 5' end of the complementary sequence; the forward sequence is shown in SEQ No.5, and the complementary sequence is shown in SEQ No.6.

[0027] Furthermore, the synthesized nucleotides were annealed to form double strands using oligonucleotide annealing buffer; the annealing reaction system was: 20 μL Annealing Buffer for DNA Oligos, 20 μL forward nucleotide sequence, 20 μL complementary nucleotide sequence, and 40 μL deionized water; the annealing process was: gradient cooling in a PCR instrument, the program was: 95°C for 2 minutes; decrease by 0.1°C every 8 seconds to 25°C; and stored at 4°C.

[0028] Furthermore, the double-stranded DNA after restriction digestion and purification was connected to the pET-32a vector using T4 DNA ligase; the ligation product was transformed into competent Escherichia coli TOP10, and a single clone was obtained under ampicillin resistance screening.

[0029] Furthermore, the expression vector was transformed into Escherichia coli BL21 (E3) strain to express the protein under IPTG induction; after optimizing the IPTG induction conditions, the fusion protein of H7N9 polypeptide and TrxA could be secreted and expressed, simplifying the protein purification process.

[0030] Furthermore, the fusion protein was purified by nickel column affinity chromatography.

[0031] Furthermore, an ELISA method for detecting H7N9 virus infection and vaccine immune serum was established using the fusion protein as the coating antigen. The specific process is as follows:

[0032] (1) The fusion protein was diluted to different concentrations, including 2.5, 1, 0.5, and 0.25 μg / mL, with coating buffer, and added to a 96-well ELISA plate at 100 μL / well for coating overnight at 4°C;

[0033] (2) Discard the coating solution and wash 4 times with PBST;

[0034] (3) Add 200 μL of 5% skim milk to each well and block at 37°C for 2 h;

[0035] (4) Discard the blocking solution and wash four times with PBST;

[0036] (5) Dilute the serum to be tested 200 and 400 times with 5% skim milk, add to the microplate, 100 μL / well, and incubate at 37°C for 1 h;

[0037] (6) Discard serum and wash 4 times with PBST;

[0038] (7) Add 1:5000 diluted goat anti-chicken enzyme-labeled secondary antibody, 100 μL / well, and incubate at 37°C for 1 h;

[0039] (8) Discard the secondary antibody and wash four times with PBST;

[0040] (9) Add 100 μL of TMB colorimetric solution to each well and incubate at room temperature for 15 min in the dark. Add 50 μL of sulfuric acid solution to terminate color development.

[0041] (10) Place the 96-well plate in an ELISA reader and read the results at a wavelength of 450 nm.

[0042] Furthermore, the OD450 value of the ELISA experiment was used to determine whether the poultry to be tested was an animal infected with the H7N9 subtype avian influenza virus.

[0043] Furthermore, when the recombinant protein coating concentration is 0.5 μg / mL and the test serum dilution is 1:200, OD450=0.25 is the critical value for determining animals infected with the H7N9 virus; when the OD450 value is ≥0.25, the poultry to be tested is an animal infected with the H7N9 subtype avian influenza virus; when OD450 is <0.25, the poultry to be tested is an animal immune to the H7N9 subtype avian influenza virus or a negative animal.

[0044] The present invention also provides an ELISA kit for detecting H7N9 subtype avian influenza virus, using the above-mentioned recombinant protein as a coating antigen.

[0045] Furthermore, the coating concentration of the above-mentioned recombinant protein is greater than 0.5 μg / mL.

[0046] The present invention also provides the use of the above-mentioned recombinant protein for normalized monitoring of H7N9 subtype avian influenza virus in poultry farms or the preparation method in the preparation of H7N9 subtype avian influenza virus detection reagents.

[0047] Furthermore, the detection reagent determines whether the poultry to be tested is infected with the H7N9 subtype avian influenza virus based on the OD450 value of the poultry serum ELISA experiment.

[0048] Furthermore, when the recombinant protein coating concentration is 0.5 μg / mL and the test serum dilution is 1:200, when the OD450 value is ≥0.25, the test poultry is an animal infected with the H7N9 subtype avian influenza virus; when the OD450 is <0.25, the test poultry is an animal immune to the H7N9 subtype avian influenza virus or a negative animal.

[0049] Beneficial Effects

[0050] The recombinant protein of the present invention comprises H7N9 HA protein polypeptide, Escherichia coli TrxA protein and His tag. In Escherichia coli, the most commonly used fusion tags are GST, MBP, TrxA, etc. to increase the solubility of the expressed protein. However, if the antigen epitope is to be expressed, the effect of the fusion tag on the conformation and epitope display of the antigen should be fully considered and evaluated. The molecular weight of tags such as GST and MBP is large (about 40-55kDa), which may affect the conformation of the fusion protein; while the molecular weight of TrxA is only about 11kDa, and it has the function of a molecular chaperone, which can maintain the normal conformation of the antigen polypeptide and the display of the epitope. In order to facilitate the purification of the fusion protein, the most commonly used His tag is added, and a low-cost nickel column can be used for purification. Therefore, considering the conformation, epitope display and purification cost of the HA polypeptide, the present invention uses the structure of TrxA-HA polypeptide-His tag, which can not only maintain the normal display of the polypeptide conformation and epitope, but also facilitate the purification of the protein.

[0051] The detection cost of virus infection by using the recombinant protein of the present invention is reduced by at least 100 times. The present invention uses H7N9 virus infection detection technology and synthetic polypeptide as antigen. The market price of polypeptide synthesis is calculated (0.35 yuan / μg). The amount of antigen used to detect a single serum is 3μg, and the antigen cost of a single serum sample is 1.05 yuan in total; the fusion protein provided by the present invention is used as an antigen. The cost of protein prokaryotic expression is calculated (the cost of raw materials such as culture medium, reagents, and consumables is calculated, 0.07 yuan / μg). The amount of antigen used to detect a single serum is 0.15μg, and the antigen cost of a single serum sample is 0.01 yuan in total. Therefore, compared with the existing method, the detection method provided by the present invention has a 100-fold reduction in antigen cost. The low detection cost is more suitable for industrialized, standardized production and large-scale detection, and is also more suitable for normalized virus detection in poultry farms.

[0052] The sensitivity of the detection method using the recombinant protein of the present invention is at least 2 times higher. Compared with the existing detection technology (critical value: OD450 = 0.5), the critical value of the detection method using the fusion protein described in this patent as the antigen is OD450 = 0.25, which is lower, and the sensitivity is at least 2 times higher.

[0053] Compared with existing chemically synthesized peptides, the preparation cycle of fusion proteins expressed by prokaryotic expression systems is shorter and the source is more stable. Chemically synthesized peptides generally take 3-4 weeks, while prokaryotic expression and purification of proteins generally take 5-7 days, which greatly shortens the cycle. In addition, under the premise of having prokaryotic expression plasmids and expression and purification methods, protein expression and purification can be performed at any time as needed, and the source of antigens is more stable and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagram of the construction of a prokaryotic expression vector containing a fusion protein gene.

[0055] Figure 2 This is the protein electrophoresis diagram of the fusion protein expressed in small amounts.

[0056] Figure 3 This is the electrophoresis diagram of large-scale expression and purification of the fusion protein.

[0057] Figure 4 This is an immunoblot image of the reactivity test of the fusion protein with H7N9 serum.

[0058] Figure 5 The figures are ELISA reaction diagrams for detecting H7N9 serum using different concentrations of fusion protein as antigen; Figure A shows the result when the protein is coated at 2.5μg / mL; Figure B shows the result when the protein is coated at 1μg / mL; Figure C shows the result when the protein is coated at 0.5μg / mL; and Figure D shows the result when the protein is coated at 0.25μg / mL.

[0059] Figure 6 This is an antigen cost estimation chart for H7N9 virus infection detection methods using peptides or fusion proteins as antigens. DETAILED DESCRIPTION

[0060] Example 1 Construction of prokaryotic expression vector for expressing H7N9 polypeptide recombinant protein

[0061] The H7N9 DIVA polypeptide sequence (sequence KNVPEIPKGRG) was used to locate the nucleotide sequence corresponding to the polypeptide amino acid on the hemagglutinin gene of the H7N9 subtype avian influenza virus. The nucleotides corresponding to the polypeptide and its complementary sequence were synthesized, and an EcoRI site was added to the 5' end of the forward sequence, and an XhoI site was added to the 5' end of the complementary sequence. The forward nucleotide sequence is:

[0062] 5-GAATTCAAGAATGTTCCTGAGATTCCAAAGGGAAGAGGCCTCGAG-3 (SEQ No. 5), the complementary nucleotide sequence is

[0063] 5-CTCGAGGCCTCTTCCCTTTGGAATCTCAGGAACATTCTTGAATTC-3 (SEQ No. 6).

[0064] Use the oligonucleotide annealing buffer (Annealing Buffer for DNAOligos) to anneal the synthesized nucleotides to form a double strand. The annealing reaction system is: 20μL Annealing Buffer for DNAOligos, 20μL forward nucleotide sequence, 20μL complementary nucleotide sequence, and 40μL deionized water. Perform a gradient cooling in the PCR instrument, and the program is: 95℃ for 2 minutes; decrease 0.1℃ every 8 seconds to 25℃; store at 4℃.

[0065] After the annealing reaction, purify the double-stranded DNA product with a DNA recovery kit. Use EcoRI and XhoI to simultaneously digest the recovered double-stranded DNA and pET-32a vector. The double-stranded DNA after digestion does not need to be electrophoresed, and can be directly purified with a DNA recovery kit; the pET-32a vector after digestion is subjected to agarose electrophoresis to recover the target fragment. Connect the digested and purified double-stranded DNA to the pET-32a vector with T4 DNA ligase. The ligation product is transformed into competent Escherichia coli TOP10, and a single clone is obtained under ampicillin resistance screening. Pick a single clone, expand the culture, extract the plasmid with a plasmid mini-extraction kit, and perform sequencing analysis after digestion identification.

[0066] The results showed that the gene sequence encoding H7N9 DIVA polypeptide was successfully cloned into the prokaryotic expression vector pET-32a. The gene expression frame mainly included TrxA protein, DIVA polypeptide and His tag encoding gene (SEQ NO.4), and the translation produced TrxA-HA polypeptide-His tag fusion protein ( Figure 1 ).

[0067] Example 2 Expression and purification of H7N9 polypeptide-recombinant fusion protein in Escherichia coli

[0068] First, we conducted a small test on the expression conditions of the polypeptide-recombinant fusion protein. The recombinant expression plasmid was transformed into the E. coli BL21 (E3) strain, and a single colony was selected for induction under different conditions, including: induction with 1.0mM IPTG at 37℃ for 4h; induction with 1.0mM IPTG at 20℃ overnight. A small amount of bacterial solution was taken and the expression of the recombinant protein was detected by SDS-PAGE.

[0069] After determining the expression conditions of the polypeptide recombinant fusion protein, the culture volume of the recombinant bacteria was enlarged to 300 mL, and after IPTG induction, the culture supernatant and the bacteria were taken to detect the expression of the target protein. Then, the target protein was purified using a nickel column.

[0070] The results of the small test showed that only a small amount of the target protein was expressed in the sample before induction, and the molecular weight of the target protein was about 26kDa; the expression of the target protein increased when induced at 37℃ for 4h with 1.0mM IPTG; the expression of the target protein was the highest when induced at 20℃ overnight with 1.0mM IPTG ( Figure 2 ). Therefore, the expression conditions of H7N9 polypeptide-recombinant protein were 1.0 mM IPTG and induced at 20°C overnight.

[0071] The results of amplification culture showed that the target protein was expressed in both the supernatant and inclusion bodies of bacterial lysate, and the expression level in the supernatant was higher than that in the inclusion bodies ( Figure 3 The target protein can be purified from the culture supernatant using nickel column affinity chromatography with a purity greater than 95% ( Figure 3 ). A total of 6.6 mg of the target protein was purified from 300 mL of bacterial supernatant.

[0072] Example 3 Effect of TrxA on the immunoreactivity of HA polypeptide

[0073] In order to evaluate the effect of TrxA on the immunoreactivity of HA polypeptide in fusion protein, the reactivity of purified fusion protein with H7N9 serum was detected by immunoblotting. If the serum can react with the fusion polypeptide normally, it means that the addition of TrxA does not affect the epitope display and immunoreactivity of HA polypeptide. Take 10 μg of purified fusion protein for SDS-PAGE electrophoresis, then transfer to PVDF membrane, use H7N9 virus infected serum as primary antibody and goat anti-chicken enzyme-labeled antibody as secondary antibody to determine the reactivity of serum to fusion protein. At the same time, the His tag of fusion protein is detected as a positive control.

[0074] The results showed that the fusion protein band could be detected by both H7N9 virus infected serum and His monoclonal antibody, and the protein molecular weight was correct (~26kDa) ( Figure 4 ), indicating that the H7N9 antibody can normally recognize the HA polypeptide epitope in the fusion protein, and the addition of TrxA has no effect on the display and immune reactivity of the HA polypeptide epitope. This indicates that the use of TrxA as a fusion tag for the HA polypeptide does not affect the correct display of the HA polypeptide antigen and the normal reaction of the serum.

[0075] Example 4 Establishment of a method for detecting H7N9 virus infection using a recombinant protein with a higher coating concentration as an antigen

[0076] We first coated the polypeptide recombinant protein at a higher concentration (2.5 and 1 μg / mL) to a microplate to establish a method for detecting H7N9 virus infection. The serum samples to be tested include: H7N9 infected serum 1 (referring to the serum of chickens infected with H7N9GD15 strain), H7N9 infected serum 2 (referring to the serum of chickens infected with H7N9 AH271 strain), H7N9 immune-challenge serum (referring to the serum of chickens immunized with H7N9 inactivated vaccine after challenge), H7N9 immune serum (referring to the serum of chickens immunized with commercial vaccine H5+H7), and negative control serum (referring to the serum of non-immunized and non-infected chickens). The ELISA operation process is:

[0077] (1) Dilute the recombinant protein to 2.5 and 1 μg / mL with coating buffer, add 100 μL / well to a 96-well ELISA plate, and coat overnight at 4°C;

[0078] (2) Discard the coating solution and wash 4 times with PBST;

[0079] (3) Add 200 μL of 5% skim milk to each well and block at 37°C for 2 h;

[0080] (4) Discard the blocking solution and wash four times with PBST;

[0081] (5) Dilute the serum 200 and 400 times with 5% skim milk, add to the microplate, 100 μL / well, and incubate at 37°C for 1 h;

[0082] (6) Discard serum and wash four times with PBST;

[0083] (7) Add 1:5000 diluted goat anti-chicken enzyme-labeled secondary antibody, 100 μL / well, and incubate at 37°C for 1 h;

[0084] (8) Discard the secondary antibody and wash four times with PBST;

[0085] (9) Add 100 μL of TMB colorimetric solution to each well and incubate at room temperature for 15 min in the dark. Add 50 μL of sulfuric acid solution to terminate color development.

[0086] (10) Place the 96-well plate in an ELISA reader and read the results at a wavelength of 450 nm.

[0087] The results show that Figure 5As shown in A and B, the negative control serum of the two dilutions did not react with the recombinant protein, indicating that the established ELISA method has strong specificity; the serum of chickens immunized with the commercial inactivated vaccine of H7N9 did not react with the polypeptide recombinant protein at any dilution. However, the serum of chickens infected with two strains of H7N9 virus and the serum of H7N9 immune-challenge virus reacted with the recombinant protein, and the OD450 of the serum diluted 200 and 400 times was higher than 0.5. Therefore, when the recombinant protein was coated at a concentration of 2.5 and 1 μg / mL, the recombinant protein only reacted with the serum infected with H7N9 virus; OD450 = 0.5 can be used as the critical value for determining H7N9 virus infection.

[0088] Example 5 Establishment of a method for detecting H7N9 virus infection using a recombinant protein with a lower coating concentration as an antigen

[0089] In order to further reduce the amount of antigen used, the recombinant protein coating concentration was reduced to 0.5 and 0.25 μg / mL, and the ELISA experiment was performed according to the above steps.

[0090] The results are as follows Figure 5 As shown in Figure C, when the coating concentration was 0.5 μg / mL, the reactivity of the recombinant protein with different H7N9 sera (200-fold dilution) was similar to that at high coating concentrations (2.5 and 1 μg / mL): the OD450 values ​​of the H7N9 infected serum and the H7N9 immune-challenge serum reacting with the recombinant protein were both higher than 0.25. However, when the serum was diluted 400 times, only the OD450 value of the H7N9 infected serum 1 reacting with the recombinant protein was higher than 0.25. Figure 5 As shown in Figure D, when the protein coating concentration dropped to 0.25 μg / mL, although OD450 = 0.2 could distinguish virus-infected and immune sera, the OD values ​​of some infected sera, immune sera and negative control sera were very close. Therefore, the lowest coating concentration of the fusion protein was finally selected as 0.5 μg / mL, the serum sample to be tested was diluted 1:200, and OD450 = 0.25 was used as the critical value of the virus infection detection method.

[0091] Example 5 Antigen cost estimation for different H7N9 virus infection detection methods

[0092] Estimation of antigen costs for peptide antigen and fusion protein antigen based assays ( Figure 6). Calculated based on the average market price of chemically synthesized polypeptides, the polypeptide cost is about 0.35 yuan / μg. A single serum sample is processed into 3 replicates, and the amount of polypeptide used to detect a single serum is 3μg. The total antigen cost for a single serum sample based on the polypeptide method is 1.05 yuan. Using the fusion protein provided by the present invention as an antigen, the preparation of antigens in a prokaryotic expression system requires inputs such as culture medium, reagents, and consumables, and the cost is about 0.07 yuan / μg. A single serum sample is processed into 3 replicates, and the amount of antigen used to detect a single serum is 0.15μg. The total antigen cost for a single serum sample based on the fusion protein method is 0.01 yuan. Compared with the disclosed polypeptide ELISA method, the detection method using fusion protein as an antigen provided by the present invention has a antigen cost reduced by about 100 times.

[0093] In summary, through the specific embodiments described in the present invention, it is proved that the H7N9DIVA polypeptide can be efficiently expressed in the form of a fusion protein in an E. coli expression system; the expressed recombinant protein can have a strong specific reaction with the serum of animals infected with the H7N9 subtype virus at a low concentration, and has a low reactivity with the serum of animals immunized with the H7N9 vaccine and normal animals. The ELISA method established based on the recombinant protein can specifically detect animals infected with the H7N9 subtype AIV. In addition, the cost of preparing the fusion protein is significantly reduced. Therefore, the present invention provides a recombinant protein and a detection method that can be used for H7N9 virus infection detection, which can replace chemically synthesized polypeptide antigens, and has the advantages of low cost, short cycle, stable source, stable detection effect, etc. The protein antigen and the corresponding detection method provided by the present invention can be used for the normalized monitoring of H7N9 subtype avian influenza in poultry farms, which is of great significance for disease prevention and control.

[0094] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A recombinant protein for routine monitoring of H7N9 subtype avian influenza virus in poultry farms, the amino acid sequence of the recombinant protein is shown in SEQ No. 1, the recombinant protein comprises an H7N9 HA protein polypeptide, an Escherichia coli TrxA protein and a His tag, the amino acid sequence of the H7N9 HA protein polypeptide is shown in SEQ No. 2; the amino acid sequence of the Escherichia coli TrxA protein is shown in SEQ No.

3.

2. The method for preparing a recombinant protein for routine monitoring of H7N9 subtype avian influenza virus in poultry farms according to claim 1, characterized in that: The gene sequence encoding the recombinant protein according to claim 1 is shown in SEQ No.

4.

3. The method for preparing a recombinant protein for routine monitoring of H7N9 subtype avian influenza virus in poultry farms according to claim 1, characterized in that: The gene encoding the H7N9 HA protein polypeptide was cloned into the prokaryotic expression vector pET32a through the restriction sites EcoRI and XhoI.

4. The method for preparing a recombinant protein for routine monitoring of H7N9 subtype avian influenza virus in poultry farms according to claim 3, characterized in that: An EcoRI site is added to the 5' end of the forward sequence corresponding to the H7N9 HA protein polypeptide, and an XhoI site is added to the 5' end of the complementary sequence; the forward sequence is shown in SEQ No. 5, and the complementary sequence is shown in SEQ No.

6.

5. The method for preparing a recombinant protein for routine monitoring of H7N9 subtype avian influenza virus in poultry farms according to claim 3, characterized in that: The double-stranded DNA after restriction digestion and purification was connected to the pET-32a vector using T4 DNA ligase; the ligation product was transformed into competent Escherichia coli TOP10, and a single clone was obtained under ampicillin resistance screening.

6. An ELISA kit for detecting H7N9 subtype avian influenza virus, characterized in that: The recombinant protein according to claim 1 is used as the coating antigen.

7. The kit according to claim 6, characterized in that The coating concentration of the recombinant protein according to claim 1 is greater than 0.5 μg / mL.

8. Use of the recombinant protein for routine monitoring of H7N9 subtype avian influenza virus in poultry farms according to claim 1 or the preparation method according to claims 2 to 5 in the preparation of a detection reagent for H7N9 subtype avian influenza virus.

9. The use according to claim 8, characterized in that: The detection reagent determines whether the poultry to be tested is infected with the H7N9 subtype avian influenza virus according to the OD450 value of the ELISA experiment of the poultry serum to be tested.

10. The use according to claim 9, characterized in that When the recombinant protein coating concentration is 0.5 μg / mL and the test serum dilution is 1:200, when the OD450 value is ≥0.25, the test poultry is an animal infected with the H7N9 subtype avian influenza virus; when the OD450 is <0.25, the test poultry is an animal immune to the H7N9 subtype avian influenza virus or a negative animal.

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