Construction method, expression system and application of recombinant protein for preventing infectious bronchitis

Abstract

The invention is applicable to the technical field of genetic engineering and biological medicine, and provides a construction method and an expression system of recombinant protein for preventing infectious bronchitis of chicken and application of the recombinant protein. Coding DNA of multi-antigen epitope tandem protein from infectious bronchitis virus N, S1 protein is connected to obtain a fusion recombinant protein IBV: NS1 coding gene, a recombinant expression plasmid pET28a-IBV: NS1 is constructed and transferred into E. coli Rosetta (DE3) competent cells to obtain an E. coli genetic engineering expression bacterium Ec-RD-pET28a-IBV: NS1, and the fusion recombinant protein IBV: NS1 is obtained through induced expression and massive purification. And mixing with an immunologic adjuvant to prepare the subunit vaccine. The subunit vaccine has the advantages of high antigen expression quantity, long antibody maintenance time, high purity, good safety, strong immunogenicity and strong protectiveness, and can effectively prevent infectious bronchitis.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and biomedicine, and particularly relates to a method for constructing a recombinant protein for preventing infectious bronchitis in chickens, an expression system and an application thereof. Background Art

[0002] Infectious bronchitis (IB) in chickens is one of the major diseases currently seriously threatening the poultry industry. With the development of the breeding industry towards large-scale and intensive directions, infectious bronchitis in chickens frequently occurs during the poultry breeding process. This disease is an acute infectious disease caused by infectious bronchitis virus (IBV) that harms the respiratory and urogenital tracts. It is highly prevalent in chicks within 40 days of age, and the mortality rate can reach over 90%. Once a chicken flock is infected, usually 100% of them get sick, and the mortality rate ranges between 5 - 30%. As the disease progresses, it is also prone to secondary or concurrent infections with diseases such as Escherichia coli, Mycoplasma, mild influenza, and atypical Newcastle disease, leading to a further increase in the mortality rate, and the infection mechanism also becomes more complex, increasing the difficulty of preventing and controlling this disease and bringing huge economic losses to farmers.

[0003] IBV belongs to the genus Coronavirus in the family Coronaviridae. Its genome is a single-stranded positive-sense RNA without segmentation, with a full length of approximately 27.4 - 27.7 kb. The virus encodes four structural proteins: S, E, M, and N. Among them, the spike protein (S) is located on the surface envelope of the virus particle and is the main component constituting the surface spikes of IBV. This protein mediates the attachment of the virus to the host cell and its entry into the host cell. The S protein consists of two subunits: the S1 subunit (about 500 - 550 amino acids) is responsible for the recognition and binding of the virus to the host cell receptor, and the S2 subunit (630 amino acids) is responsible for the fusion of the virus with the host cell membrane, enabling the viral genome to enter the host cell. The S protein is the main antigenic component of IBV and is responsible for inducing neutralizing antibodies and protective immunity against the virus. There are immune recognition-related targets on the nucleocapsid protein (N), which mediates cellular immunity and humoral immunity. The membrane protein (M) is the most abundant structural protein and can maintain the shape of the virus. The small membrane protein (E) exists in small amounts and promotes the assembly and release of the virus from the host cell.

[0004] For the prevention of IB, vaccination is the most economical and effective means. Traditional vaccines mainly include inactivated vaccines and attenuated live vaccines. However, in the face of virus strains with numerous serotypes like IB, traditional vaccines cannot match the prevalent strains, and the protection effect is limited. In addition, the virus strains of traditional vaccines have poor stability, often showing the phenomenon of reversion to virulence, and they have high production costs, complex preparation processes, and cumbersome immunization procedures, resulting in uneven levels of commercial vaccines. Therefore, developing a new and effective vaccine has become an urgent task. For this reason, the present invention proposes a method for constructing a recombinant protein for preventing infectious bronchitis in chickens, an expression system and an application thereof. Summary of the Invention

[0005] The object of the present invention is to provide a method for constructing a recombinant protein for preventing infectious bronchitis in chickens, an expression system and an application thereof, aiming to solve the problems raised in the above-mentioned background technology.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] A recombinant protein for preventing infectious bronchitis in chickens, wherein the recombinant protein is a fusion recombinant protein IBV:NS1, and the amino acid sequence of the fusion recombinant protein IBV:NS1 is as follows:

[0008] KFEGSGVPDNENLKNSQQHGYWRRQARYKQGKGGRKPVPDAWGGGGSVKSRSN

[0009] QGTRDPDKFDQFPLRFSDGGPDGNFRWDFIPLNRGRSGRSTAAGGGGSAASSRVPSREGS

[0010] RGRRSGAGGGGSFGPRTKGKEGNGGGGSVTPKLQPDGLHGGGGSTVVPRDDPQFDNY

[0011] VKICGGGGSVGTRPKDEVVRPKSRSSSRPATRGNSPAPKGGGGSCDNSPKGLLACQYNT

[0012] GNFSDGFYPFTNSTLVREGGGGSTNVSNAQPNSGGVNTGGGGSMYGSYHPSCSFRPETI

[0013] NSGLWFGGGGSKSDGSRIQTRTEPGGGGSHNYNNIT (as shown in SEQ ID NO.1).

[0014] The nucleotide sequence of the coding gene of the fusion recombinant protein IBV:NS1 is as follows:

[0015] AAATTTGAAGGTTCTGGTGTTCCGGATAATGAAAACCTGAAAAATTCTCAGCAG

[0016] CACGGTTATTGGCGTCGTCAGGCACGTTATAAACAGGGTAAAGGTGGTCGTAAACC

[0017] GGTTCCGGATGCTTGGGGTGGTGGTGGTAGCGTTAAATCTCGTTCTAACCAGGGTAC

[0018] TCGTGATCCGGATAAATTCGACCAGTTCCCGCTGCGTTTCTCCGATGGCGGACCGGA

[0019] TGGCAACTTCCGCTGGGACTTCATCCCGCTGAACCGTGGTCGTTCTGGTCGTAGCAC

[0020] TGCGGCTGGCGGTGGTGGTAGCGCTGCGAGCAGCCGTGTTCCGAGCCGCGAAGGCA

[0021] GCCGTGGTCGTCGTTCAGGTGCGGGCGGCGGCGGCTCCTTCGGCCCGCGTACCAAA

[0022] GGCAAAGAAGGCAACGGCGGGGGCGGTTCCGTTACCCCGAAACTGCAGCCGGATGG

[0023] TCTGCACGGTGGCGGCGGCAGCACCGTGGTTCCGCGCGATGATCCGCAGTTCGATA

[0024] ACTACGTTAAAATCTGCGGCGGTGGCGGCAGCGTTGGCACCAGGCCGAAAGATGAA

[0025] GTTGTTCGTCCGAAATCTCGCAGCTCTAGCCGTCCGGCGACCCGCGGCAACAGCCCG

[0026] GCGCCGAAAGGTGGTGGTGGTAGCTGTGACAACAGCCCGAAAGGGCTGCTGGCGTG

[0027] CCAGTACAACACTGGCAACTTCAGCGATGGCTTCTACCCGTTCACCAACAGCACCCT

[0028] GGTTCGTGAAGGCGGCGGCGGCAGCACCAACGTTAGCAACGCTCAGCCGAACAGCG

[0029] GTGGCGTTAACACCGGTGGCGGCGGCAGTATGTACGGCAGCTACCACCCGAGCTGT

[0030] AGCTTCCGTCCGGAAACCATTAACTCTGGTCTGTGGTTCGGTGGCGGTGGCTCTAAA

[0031] TCTGATGGTAGCCGTATCCAAACCCGTACCGAACCGGGTGGCGGCGGTAGCCATAA

[0032] CTATAACAACATCACC (as shown in SEQ ID NO.2).

[0033] A method for constructing a coding gene of the recombinant protein described above, comprising the following steps:

[0034] Using the restriction endonuclease digestion site ligation method and the Overlap PCR ligation method through a flexible linker, the coding DNA of the multi-antigen epitope tandem protein derived from the N protein of QX-type infectious bronchitis virus (IBV:N) and the coding DNA of the multi-antigen epitope tandem protein derived from the S1 protein of QX-type infectious bronchitis virus (IBV:S1) are tandemly connected to obtain the coding gene of the fusion recombinant protein IBV:NS1.

[0035] Furthermore, the coding DNA of the multi-antigen epitope tandem protein derived from the N protein of QX-type infectious bronchitis virus includes gene fragments of multiple antigen epitopes, which are tandemly spaced by a flexible linker, and chemically synthesize the codon-optimized coding DNAIBV:N, as shown in 1-627bp of SEQ ID NO.2, and the corresponding amino acid sequence is as shown in 1-209aa of SEQ ID NO.1;

[0036] The coding DNA of the multi-antigen epitope tandem protein derived from the S1 protein of QX-type infectious bronchitis virus includes gene fragments of multiple antigen epitopes, which are tandemly spaced by a flexible linker, and chemically synthesize the codon-optimized coding DNAIBV:S1, as shown in 643-972bp of SEQ ID NO.2, and the corresponding amino acid sequence is as shown in 215-324aa of SEQ ID NO.1.

[0037] Furthermore, the restriction endonuclease digestion site ligation method is to introduce the restriction endonuclease digestion sites of NcoI, EcoRI, HindIII, and XhoI into the upstream primers and downstream primers of the coding DNA of the multi-antigen epitope tandem proteins of IBV:N and IBV:S1;

[0038] The Overlap PCR method is to introduce overlapping regions of DNA sequences into the upstream and downstream primers of the coding DNA of the multi-epitope tandem protein, including:

[0039] The upstream primer of the coding DNA of the multi-epitope tandem protein derived from the N protein of QX-type infectious bronchitis virus IBV:N contains three restriction enzyme sites, NcoI, EcoRI, and HindIII, as shown in SEQ ID NO.3; the downstream primer contains a partial overlapping sequence of IBV:S1, as shown in SEQ ID NO.4.

[0040] The upstream primer of the coding DNA of the multi-epitope tandem protein derived from the S1 protein of QX-type infectious bronchitis virus IBV:S1 contains a partial overlapping sequence of IBV:N, as shown in SEQ ID NO.5; the downstream primer contains a XhoI restriction enzyme site, as shown in SEQ ID NO.6.

[0041] A recombinant expression system for the recombinant protein described above, the recombinant expression system includes a eukaryotic expression system or a prokaryotic expression system.

[0042] A recombinant expression plasmid for expressing the recombinant protein described above, the basic vector of the recombinant expression plasmid includes pET-28a. The upstream primer of the fusion recombinant protein coding DNA IBV:NS1 is introduced with three restriction enzyme sites, NcoI, EcoRI, and HindIII, as shown in SEQ ID NO.3, and the downstream primer is introduced with a linker and a XhoI restriction enzyme site, as shown in SEQ ID NO.6. It is inserted between the NcoI and XhoI restriction enzyme sites of pET-28a by double digestion and ligation to construct a recombinant expression plasmid pET28a-IBV:NS1 for the fusion recombinant protein IBV:NS1.

[0043] An induction expression method for the recombinant protein described above, including the following steps: transferring the recombinant expression plasmid pET28a-IBV:NS1 into Escherichia coli E.coli Rosetta(DE3) competent cells to obtain an Escherichia coli genetic engineering expression strain Ec-RD-pET28a-IBV:NS1 that can express the fusion recombinant protein IBV:NS1, and using IPTG induction expression and inclusion body renaturation methods to obtain the fusion recombinant protein IBV:NS1.

[0044] An application of the recombinant protein described above or the recombinant protein obtained by using the induction expression method described above in the preparation of a subunit vaccine against infectious bronchitis in chickens.

[0045] An infectious bronchitis subunit vaccine for chickens, said subunit vaccine comprising an immune adjuvant and the recombinant protein as described above or a recombinant protein obtained by using the induction expression method as described above.

[0046] Furthermore, the immune adjuvant and the recombinant protein are mixed at a volume ratio of 1:1, and the immune adjuvant is an aluminum hydroxide sol adjuvant.

[0047] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0048] The present invention selects multiple antigenic epitopes of the infectious bronchitis virus capsid protein and constructs a fusion recombinant protein IBV:NS1. The infectious bronchitis subunit vaccine for chickens prepared by using this protein can stimulate the body to produce more types and quantities of protective antibodies due to the rich antigenic epitopes of the capsid protein, and significantly improve the protection against infectious bronchitis in chickens. At the same time, the selected multiple antigenic epitopes and the optimization of the codons of the DNA encoding the fusion recombinant protein IBV:NS1 according to the codon preference of Escherichia coli significantly improve the expression level of the fusion recombinant protein IBV:NS1 in the Escherichia coli expression system, ensuring sufficient fusion recombinant protein IBV:NS1 for vaccine preparation. In summary, the subunit vaccine based on the fusion recombinant protein IBV:NS1 has the advantages of high antigen expression level, long antibody maintenance time, high purity, good safety, strong immunogenicity, strong protection, etc., and can effectively prevent infectious bronchitis in chickens. Description of the Drawings

[0049] Figure 1 It is a PCR amplification result diagram of the fusion recombinant protein coding gene IBV:NS1 obtained by connecting the DNA XCIBV:N and XCIBV:S1 encoding two multiple antigenic epitope tandem proteins in step (2) of Example 1 of the present invention by the Overlap PCR method; wherein: lane M is DL 5000 DNA Marker; lane 1 is the coding gene IBV:NS1 of the fusion recombinant protein IBV:NS1.

[0050] Figure 2It is the agarose gel electrophoresis result diagram of the double digestion products after the recombinant cloning plasmid pMD19-T-IBV:NS1 inserting the fusion recombinant protein coding gene and the pET-28a vector in Example 2 of the present invention were identified by double digestion with restriction endonucleases NcoI and XhoI; wherein: the first lane M is DL 5000 DNA Marker; the second lane M is DL 15000 DNA Marker; lane 1 is the fusion recombinant protein coding gene IBV:NS1 after the recombinant cloning plasmid pMD19-T-IBV:NS1 was double digested; lane 2 is the fusion recombinant protein coding gene IBV:NS1 without digestion; lane 3 is the product NcoI-pET28a-XhoI after the pET-28a vector was double digested; lane 4 is the pET-28a vector without digestion.

[0051] Figure 3 It is the PCR amplification result diagram of the coding gene of the fusion recombinant protein IBV:NS1 obtained after double digestion and ligation of the pMD19-T IBV:NS1 and the pET-28a vector in Example 2 of the present invention; wherein: lane M is DL5000 DNA Marker; lane 1 is the coding gene IBV:NS1 after double digestion and ligation.

[0052] Figure 4 It is the SDS-PAGE electrophoresis detection result diagram of the fusion recombinant protein IBV:NS1 in step (1) of Example 3 of the present invention; wherein: lane M is 10-250 kDa Marker; lane 1 is the total protein of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 without induced expression; lane 2 is the total protein of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 with induced expression; lane 3 is the total supernatant protein of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 without induced expression; lane 4 is the total supernatant protein of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 with induced expression; lane 5 is the total precipitated protein of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 with induced expression; lane 6 is the total precipitated protein of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 without induced expression.

[0053] Figure 5 It is the SDS-PAGE electrophoresis detection result diagram of the purified fusion recombinant protein IBV:NS1 in step (2) of Example 3 of the present invention; wherein: lane M is 10-180 kDa Marker; lane 1 is the pET28a empty vector; lane 2 is the total precipitated protein of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 with induced expression; lane 3 is the purified fusion recombinant protein IBV:NS1 of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 with induced expression.

[0054] Figure 6 It is the graph of the change level of chicken serum antibody after protein immunization in step (2) of Example 4 of the present invention;

[0055] Figure 7 It is the survival curve graph of chickens after two immunizations and challenge experiments in the immunization challenge protection experiment in step (2) of Example 4 of the present invention. Detailed implementation manners

[0056] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention will be described in detail below, but it should not be construed as a limitation on the implementable scope of the present invention.

[0057] The following describes the specific implementation of the present invention in detail in combination with specific embodiments.

[0058] Example 1: Construction of a cloning vector for the fusion recombinant protein IBV:NS1 coding gene;

[0059] (1) Antigenicity prediction was performed on the amino acid sequences of two capsid proteins N and S1 of the ck / CH / LJS / 101109 strain (GenBank accession number KX219794.1) of the QX genotype of infectious bronchitis virus (IBV). According to the characteristics of antigenic epitopes, protein fragments located in the extracellular region, with good hydrophilicity and concentrated antigenic epitopes were selected, and the coding DNA nucleotide sequences of the protein flexible linker GGGGS were used to ligate the coding DNAs of the selected protein fragments with concentrated antigenic epitopes to construct two multi-antigenic epitope tandem protein coding DNAs, namely IBV:N and IBV:S1. The two multi-antigenic epitope tandem protein coding DNAs were directly synthesized by chemical methods after codon optimization according to the codon usage preference of the Escherichia coli expression system. The DNA synthesis was completed by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.

[0060] (2) Using the Overlap PCR method, IBV:N and IBV:S1 were serially connected in the order of N-S1. SnapGene was used to design the Overlap primers for IBV:N and IBV:S1. Using the multi-antigenic epitope tandem protein coding DNAs IBV:N and IBV:S1 as templates, IBV:N and IBV:S1 with sticky ends at the NcoI and XhoI restriction enzyme cleavage sites were amplified. Using the primers in Table 1 and the PCR amplification system and conditions in Table 2 for PCR amplification, the coding gene of the fusion recombinant protein IBV:NS1 with different restriction enzyme cleavage sites at both the 5' and 3' ends was obtained. The specific steps are as follows:

[0061] Using the coding DNA of the multi-epitope tandem protein IBV:N as a template, PCR amplification was carried out with the upstream primer XCN:NS1-F containing NcoI, EcoRI, and HindIII restriction enzyme sites (shown in SEQ ID NO.3) and the downstream primer XCN:NS1-R containing a partial IBV:S1 homologous sequence (shown in SEQ ID NO.4) to amplify the DNA fragment XCIBV:N. Using the coding DNA of the multi-epitope tandem protein IBV:S1 as a template, PCR amplification was carried out with the upstream primer XCS1:NS1-F containing a partial IBV:N homologous sequence (shown in SEQ ID NO.5) and the downstream primer XCS1:NS1-R containing an XhoI restriction enzyme site (shown in SEQ ID NO.6) to amplify the DNA fragment XCIBV:S1. Using XCIBV:N and XCIBV:S1 as templates, PCR amplification was carried out with the upstream primer XCN:NS1-F containing NcoI, EcoRI, and HindIII restriction enzyme sites (shown in SEQ ID NO.3) and the downstream primer XCS1:NS1-R containing a linker and an XhoI restriction enzyme site (shown in SEQ ID NO.6). The amplification product was a DNA fragment with a length greater than 1000bp. After the PCR reaction, the PCR product was subjected to 1% agarose gel electrophoresis. The results were as Figure 1 shown, with a clear and bright band at greater than 1000bp. Then, gel extraction was performed. Using a T-vector ligation kit, the recovered DNA fragment was ligated with the pMD-19T(Simple) vector overnight in a 16°C water bath. The ligation system is shown in Table 3.

[0062] Table 1 Information of all primers

[0063]

[0064]

[0065] Table 2 PCR amplification system and conditions

[0066]

[0067] Note: The PCR reaction program was 98°C for 5 min; 35 cycles (98°C for 15 s; 60°C for 40 s; 72°C for 30 s); 72°C for 5 min; stored at 4°C.

[0068] Table 3 Ligation system

[0069]

[0070] (3) The recombinant cloning plasmid containing the target DNA obtained after the connection in step (2) was transformed into the Escherichia coli competent cell E. coli DH5α, and the positive transformants were picked and sent to Sangon Biotech Co., Ltd. for sequencing.

[0071] Example 2: Construction of a prokaryotic expression genetic engineering bacterium of the fusion recombinant protein IBV:NS1;

[0072] The recombinant cloning plasmid pMD19-T-IBV:NS1 inserted with the target DNA fragment was extracted using a plasmid extraction kit, and the coding gene of the fusion recombinant protein was constructed by using the restriction endonuclease digestion site ligation method. The specific steps are as follows:

[0073] The recombinant cloning plasmid pMD19-T-IBV:NS1 was double-digested with NcoI and XhoI, and the system is shown in Table 4; the pET-28a vector was double-digested with NcoI and XhoI, and the system is shown in Table 5. The system was incubated at 37 °C for 1 h for digestion. After the digestion was completed, the digestion products were subjected to 1% agarose gel electrophoresis, and the results are as Figure 2 shown. After double-digestion of the recombinant cloning plasmid pMD19-T-IBV:NS1, there were 2 bands, the size was consistent with the expectation, and there were no non-specific bands; after double-digestion of the pET-28a vector, there was 1 band, the size was consistent with the expectation, and there were no non-specific bands. The target DNA fragment was recovered from the gel using a DNA gel recovery kit to obtain the target DNA fragment with sticky ends of different restriction sites, and it was ligated with T4 DNA ligase to obtain the coding gene of the fusion recombinant protein. The ligation system (shown in Table 6) was placed in a 16 °C constant temperature water bath for overnight ligation, and the ligation product was transformed into Escherichia coli E. coli Rosetta(DE3). The positive clone bacteria successfully transfected with the recombinant expression plasmid were identified by PCR. The PCR amplification results of the coding gene of the fusion recombinant protein IBV:NS1 are as Figure 3 shown, and there was a clear and bright single band at greater than 1000 bp. The selected positive clone bacteria were sent to a biological company for sequencing. The plasmid with correct sequencing results was the prokaryotic expression plasmid pET28a-IBV:NS1 for expressing the fusion recombinant protein IBV:NS1, and the E. coli Rosetta(DE3) expression bacteria containing this recombinant expression plasmid were named Ec-RD-pET28a-IBV:NS1.

[0074] Table 4 Double-digestion system of the recombinant cloning plasmid pMD19-T-IBV:NS1

[0075]

[0076] Table 5 Double-digestion system of the pET-28a vector

[0077]

[0078]

[0079] Table 6 Target Fragment Ligation System

[0080]

[0081] Example 3: Expression and Purification of the Fusion Recombinant Protein IBV:NS1

[0082] (1) Inoculate the genetically engineered expression strain Ec-RD-pET28a-IBV:NS1 into 5 mL of liquid LB medium containing 100 μg / mL kanamycin, culture at 37 °C for 2 h, then add IPTG to a final concentration of 0.5 mmol / L and induce for 8 h. Verify the successful expression of the target fusion recombinant protein IBV:NS1 by SDS-PAGE electrophoresis. Ultrasonically disrupt the induced expression strain, centrifuge the sonicated bacterial solution at 8000 rpm and 4 °C for 10 min, then collect the supernatant and precipitate, and detect the expression level and soluble state of the target protein IBV:NS1 in the genetically engineered expression strain by SDS-PAGE electrophoresis.

[0083] The SDS-PAGE electrophoresis detection results of the genetically engineered expression strain Ec-RD-pET28a-IBV:NS1 are as Figure 4 shown. It can be found from the figure that the fusion recombinant protein IBV:NS1 can be successfully induced to express, and the total protein of the precipitate of the genetically engineered expression strain Ec-RD-pET28a-IBV:NS1 induced for expression is good, indicating that the main expression form of the protein is inclusion body expression.

[0084] (2) Renaturation of the fusion recombinant protein IBV:NS1

[0085] After inducing the expression of the genetically engineered expression strain Ec-RD-pET28a-IBV:NS1 with IPTG, collect the bacterial cell precipitate. In view of the SDS-PAGE detection result showing that the recombinant protein is expressed in inclusion bodies, the inclusion body renaturation and purification method is used to purify the recombinant protein. The specific operation is as follows:

[0086] ① Scale-up culture and induction of expression: Inoculate 5 mL of the Ec-RD-pET28a-IBV:NS1 bacterial solution cultured overnight at 37 °C into 1 L of sterilized LB medium, add 100 μg / mL kanamycin, culture at 37 °C and 180 rpm on a shaker for 2 h, then add IPTG to a final concentration of 0.5 mmol / L, and continue to induce on a shaker at 37 °C and 180 rpm for 8 h. After the induction is completed, centrifuge the bacterial solution at 8000 rpm and 4 °C for 10 min to collect the bacterial cell precipitate.

[0087] ② Bacterial cell washing and inclusion body collection: Wash the collected bacterial cells twice with 1×PBS, centrifuge at 8000 rpm and 4°C for 10 min, and finally collect the bacterial cell pellet.

[0088] Suspend the bacterial cells in TE buffer (TE buffer formulation: 2.4228 g of Tris, 0.2923 g of EDTA, made up to 1 L, pH adjusted to 8.5) at a ratio of 1:100 (bacterial cell wet weight to TE buffer volume), and then perform ultrasonic disruption ( 10% power, ultrasonic treatment for 3 s, pause for 5 s, for a total of 40 min). After disruption, centrifuge at 8000 rpm and 4°C for 40 min to collect the inclusion bodies.

[0089] ③ Inclusion body washing:

[0090] Add washing buffer I at a ratio of 1:50 (g / mL) (inclusion body wet weight to washing buffer I volume) (washing buffer I formulation: 2.4228 g of Tris, 0.2923 g of EDTA, 10 mL of Tritonx-100, made up to 1 L), wash at 200 rpm at room temperature for 2 h, centrifuge at 8000 rpm and 4°C for 40 min, and collect the inclusion body pellet. Repeat this washing step once.

[0091] Add the collected inclusion body pellet to washing buffer II at a ratio of 1:50 (g / mL) (inclusion body wet weight to washing buffer II volume) (washing buffer II formulation: 2.4228 g of Tris, 0.2923 g of EDTA, 10 mL of Tritonx-100, 120.12 g of Urea, made up to 1 L, pH adjusted to 8.5), wash at 200 rpm at room temperature for 2 h, centrifuge at 8000 rpm and 4°C for 40 min, and collect the inclusion body pellet. Repeat this washing step once.

[0092] ④ Inclusion body denaturation: Dissolve the washed inclusion bodies in denaturing buffer at a ratio of 1:15 (denaturing buffer formulation: 2.4228 g of Tris, 2.923 g of EDTA, 480.48 g of Urea, made up to 1 L, pH adjusted to 9.5), stir overnight at 4°C and 120 rpm, and then centrifuge at 8000 rpm and 4°C for 40 min. The supernatant obtained is the denatured inclusion body.

[0093] ⑤ Renaturation of denatured protein: Pour the denatured protein into the treated dialysis bag. According to the volume ratio of denaturing solution to renaturation buffer of 1:100, immerse the dialysis bag containing the denatured protein successively into renaturation buffer I (2.4228 g of Tris, 0.61464 g of GSH, 0.122526 g of GSSG, 360.36 g of Urea, made up to 1 L, adjusted to pH 9.5), renaturation buffer II (2.4228 g of Tris, 0.61464 g of GSH, 0.122526 g of GSSG, 240.24 g of Urea, made up to 1 L, adjusted to pH 9.5), renaturation buffer III (2.4228 g of Tris, 0.61464 g of GSH, 0.122526 g of GSSG, 120.12 g of Urea, made up to 1 L, adjusted to pH 9.5), and renaturation buffer IV (2.4228 g of Tris, 0.61464 g of GSH, 0.122526 g of GSSG, made up to 1 L, adjusted to pH 9.5) for renaturation. The renaturation system is placed in a 4°C dialysis cabinet and stirred very slowly, with each dialysis for 6 - 12 h. Finally, dialyze the denaturing solution into PBS buffer with a pH of 7.4, centrifuge at 8000 rpm and 4°C for 30 min, take the supernatant, and filter and sterilize it with a 0.22 nm filter membrane.

[0094] ⑥ Protein concentration determination and detection: Use the BCA protein concentration determination kit from Beyotime Institute of Biotechnology to determine the concentration of the purified target protein. After determination, the concentration of the purified fusion recombinant protein IBV:NS1 is 1 mg / mL. The SDS-PAGE electrophoresis detection results of the purified fusion recombinant protein IBV:NS1 are as Figure 5 shown, and there is a target protein band at the expected size for the purified fusion recombinant protein IBV:NS1.

[0095] Example 4: Preparation and immunoprotective analysis experiment of a genetically engineered subunit vaccine against infectious bronchitis in chickens;

[0096] (1) Preparation of the subunit vaccine;

[0097] Dilute the purified fusion recombinant protein IBV:NS1 obtained in Example 3 to 100 μg / mL and mix it evenly with aluminum hydroxide sol adjuvant at a volume ratio of 1:1 to prepare the subunit vaccine.

[0098] (2) Immunogenicity analysis experiment of the subunit vaccine;

[0099] ① Immunogenicity analysis;

[0100] Fourteen-day-old specific pathogen-free (SPF) Hy-Line White laying hens were selected and divided into two groups of six each. One group was the immunized group, which was immunized with a subunit vaccine prepared with the fusion recombinant protein IBV:NS1; the other group was the PBS control group, which was injected with the same volume of PBS. The adjuvant was aluminum hydroxide sol, the immunization period was 14 days, the immunization program was divided into two immunizations, and the immunization method was subcutaneous injection in the neck. Starting one week after the first immunization of the chicken flock, serum samples of the immunized chickens were collected by wing vein blood collection every week for detecting the change level of antibody titer. The antibody titer in the serum was detected by the indirect ELISA method and the P / N value was calculated. The results are as Figure 6 shown. It can be seen from the results that the subunit vaccine prepared with the fusion recombinant protein IBV:NS1 can stimulate the body to produce antibodies, and the antibody maintenance time is relatively long. This indicates that the prepared fusion recombinant protein IBV:NS1 has good immunogenicity, and the vaccine composed of it and the adjuvant can stimulate the body to produce antibodies with strong binding ability and long maintenance time.

[0101] ② Animal challenge immunoprotection experiment;

[0102] A. Grouping and immunization of experimental chickens;

[0103] Fourteen-day-old SPF Hy-Line White laying hens were selected, with six in each group. The immunization period was 14 days, the immunization program was divided into two immunizations, and the immunization method was subcutaneous injection in the neck. The specific grouping is as follows:

[0104] Immunization experimental group: Two-immunization group. At 14 days of age, the chickens were immunized with the above-prepared subunit vaccine (100 μg protein / chicken), and challenged after 14 days of immunization;

[0105] Healthy blank control group: Non-immunized and non-challenged group;

[0106] Challenge control group: Challenged group injected with PBS.

[0107] B. Challenge immunoprotection experiment;

[0108] After the second immunization, the immunization experimental group and the challenge control group of chickens were subcutaneously injected in the neck with the M41 isolate and challenged at a dose of 100 TCID 50 . One week after the challenge, the clinical symptoms of the chickens were observed every day, the death situation of the infected chickens was recorded, the dead chickens were necropsied, and the lesions were observed. The results of the challenge immunoprotection experiment are as Figure 7 shown. It can be seen that all chickens in the immunization experimental group did not get sick, the mortality rate after challenge was 0%, and the challenge protection rate was 100%; all chickens in the challenge control group got sick and died after challenge; no chickens in the healthy blank control group got sick or died. The results indicate that the subunit vaccine prepared with the fusion recombinant protein IBV:NS1 has complete protective effects, and two immunizations can provide 100% immunoprotection rate.

[0109] Conclusion: The fusion recombinant protein IBV:NS1 of the present invention has good immunogenicity, and the subunit vaccine composed of it and adjuvant can stimulate the body to produce antibodies with strong binding ability and long maintenance time. The subunit vaccine prepared from the fusion recombinant protein IBV:NS1 in the present invention can exert a complete protective effect only after two immunizations, significantly reducing the immunization cost, indicating that the subunit vaccine prepared from the fusion recombinant protein IBV:NS1 is both efficient and economical.

[0110] The above are only the preferred embodiments of the present invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent.

Claims

1. A recombinant protein for preventing infectious bronchitis in chickens, characterized in that: The recombinant protein is a fusion recombinant protein IBV:NS1, the amino acid sequence of the fusion recombinant protein IBV:NS1 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the fusion recombinant protein IBV:NS1 is shown in SEQ ID NO.

2.

2. A method for constructing a gene encoding a recombinant protein according to claim 1, characterized in that: The following steps are involved: Using the restriction endonuclease site connection method and the Overlap PCR connection method, the encoding DNA IBV:N of the multi-epitope tandem protein derived from the QX type infectious bronchitis virus N protein and the encoding DNA IBV:S1 of the multi-epitope tandem protein derived from the QX type infectious bronchitis virus S1 protein were concatenated through a flexible linker to obtain the encoding gene of the fusion recombinant protein IBV:NS1.

3. The construction method according to claim 2, characterized in that: The coding DNA of the multi-epitope tandem protein derived from the QX infectious bronchitis virus N protein includes gene fragments of multiple antigenic epitopes, which are tandemly connected with flexible linkers, and the coding DNA IBV:N is chemically synthesized and codon-optimized, as shown in 1-627 bp of SEQ ID NO.2, and the corresponding amino acid sequence is shown in 1-209aa of SEQ ID NO.1; The coding DNA of the multi-antigenic epitope tandem protein derived from the S1 protein of QX infectious bronchitis virus includes gene fragments of multiple antigenic epitopes, which are tandemly connected with flexible linkers, and the coding DNA IBV:S1 optimized by codons is chemically synthesized, as shown in 643-972bp of SEQ ID NO.2, and the corresponding amino acid sequence is shown in 215-324aa of SEQ ID NO.

1.

4. The construction method according to claim 2, characterized in that: The restriction endonuclease cleavage site connection method is to introduce four restriction endonuclease cleavage sites, NcoI, EcoRI, HindIII, and XhoI, into the upstream primer and downstream primer of the DNA encoding the IBV:N and IBV:S1 multi-antigen epitope tandem protein; The Overlap PCR method is to introduce the overlapping region of the DNA sequence into the upstream primer and the downstream primer of the DNA encoding the multi-epitope tandem protein, comprising: The upstream primer of the DNA IBV:N encoding the multi-epitope tandem protein derived from the QX infectious bronchitis virus N protein contains three restriction endonuclease sites: NcoI, EcoRI, and HindIII, as shown in SEQ ID NO.3; the downstream primer contains a partially overlapping sequence of IBV:S1, as shown in SEQ ID NO.4; The upstream primer of the DNA IBV:S1 encoding the multi-epitope tandem protein derived from the S1 protein of QX type infectious bronchitis virus contains a partially overlapping sequence of IBV:N, as shown in SEQ ID NO.5; the downstream primer contains an XhoI restriction endonuclease site, as shown in SEQ ID NO.

6.

5. A recombinant expression system of a recombinant protein according to claim 1, characterized in that: The recombinant expression system includes a eukaryotic expression system or a prokaryotic expression system.

6. A recombinant expression plasmid for expressing the recombinant protein according to claim 1, characterized in that: The basic vector of the recombinant expression plasmid includes pET-28a, and the upstream primer of the fusion recombinant protein encoding DNA IBV:NS1 is introduced into three restriction endonuclease sites of NcoI, EcoRI, and HindIII, as shown in SEQ ID NO.3, and the downstream primer introduces linker and XhoI restriction endonuclease sites, as shown in SEQ ID NO.6, and is inserted between the NcoI and XhoI restriction sites of pET-28a through double enzyme digestion and connection to construct the recombinant expression plasmid pET28a-IBV:NS1 of the fusion recombinant protein IBV:NS1.

7. A method for inducing expression of a recombinant protein according to claim 1, characterized in that: The following steps are involved: The recombinant expression plasmid pET28a-IBV:NS1 was transferred into competent cells of E. coli Rosetta (DE3) to obtain the E. coli genetic engineering expression bacteria Ec-RD-pET28a-IBV:NS1 that can express the fusion recombinant protein IBV:NS1, and the fusion recombinant protein IBV:NS1 was obtained by using IPTG-induced expression and inclusion body renaturation method.

8. Use of the recombinant protein according to claim 1 or the recombinant protein obtained by the induced expression method according to claim 7 in the preparation of an avian infectious bronchitis subunit vaccine.

9. A subunit vaccine for avian infectious bronchitis, characterized in that: The subunit vaccine comprises an immune adjuvant and the recombinant protein according to claim 1 or the recombinant protein obtained by the induced expression method according to claim 8.

10. The subunit vaccine according to claim 9, characterized in that The immune adjuvant and the recombinant protein are mixed in a volume ratio of 1:1, and the immune adjuvant is an aluminum hydroxide sol adjuvant.

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