A method for constructing a recombinant protein for preventing infectious bronchitis in chickens, its expression system, and its application.

Abstract

This invention relates to the fields of genetic engineering and biomedicine, and provides a method for constructing and expressing a recombinant protein for the prevention of infectious bronchitis in chickens, as well as its application. The invention involves linking the DNA encoding the multi-antigenic epitope tandem protein of the N and S1 proteins of infectious bronchitis virus (IBV) to obtain the encoding gene of the fusion recombinant protein IBV:NS1. The recombinant expression plasmid pET28a-IBV:NS1 is constructed and transformed into competent *E. coli* Rosetta (DE3) cells to obtain the genetically engineered *E. coli* expression strain Ec-RD-pET28a-IBV:NS1. After induction of expression and large-scale purification, the fusion recombinant protein IBV:NS1 is obtained and mixed with an adjuvant to prepare a subunit vaccine. This subunit vaccine exhibits high antigen expression levels, long antibody retention time, high purity, good safety, strong immunogenicity, and strong protection, effectively preventing infectious bronchitis in chickens.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biomedicine, and particularly relates to a method for constructing and expressing a recombinant protein for the prevention of infectious bronchitis in chickens, as well as its application. Background Technology

[0002] Infectious bronchitis (IB) is one of the major diseases seriously threatening the poultry industry. With the development of large-scale and intensive poultry farming, IB occurs frequently in poultry farming. This acute infectious disease, caused by the infectious bronchitis virus (IBV), damages the respiratory and urogenital tracts. It is highly prevalent in chicks under 40 days old, with a mortality rate exceeding 90%. Once infected, the flock typically shows 100% symptomatic development, with a mortality rate between 5% and 30%. As the disease progresses, it is prone to secondary or concurrent infections such as E. coli, mycoplasma, mild influenza, and atypical Newcastle disease, further increasing the mortality rate and complicating the transmission mechanism. This increases the difficulty of prevention and control, resulting in significant economic losses for poultry farmers.

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

[0004] For the prevention of infectious bronchitis (IB), vaccination is the most economical and effective means. Traditional vaccines mainly include inactivated vaccines and live attenuated vaccines. However, facing viral strains with numerous serotypes like IB, traditional vaccines cannot match the circulating strains, resulting in limited protective efficacy. Furthermore, traditional vaccines suffer from poor viral strain stability, often exhibiting virulence reversion, and are characterized by high production costs, complex preparation processes, and cumbersome immunization procedures, leading to inconsistent quality among commercially available vaccines. Therefore, developing a novel and effective vaccine is of paramount importance. To this end, this invention proposes a method for constructing and expressing a recombinant protein for the prevention of infectious bronchitis in chickens, as well as its application. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing and expressing a recombinant protein for preventing infectious bronchitis in chickens, as well as its application, in order to solve the problems mentioned in the background art.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A recombinant protein for the prevention of 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] KFEGSGVPDNENLKNSQQHGYWRRQARYKQGKGGRKPVPDAWGGGGSVKSRSNQGTRDPDKFDQFPLRFSDGGPDGNFRWDFIPLNRGRSGRSTAAGGGGSAASSRVPSREGSRGRRSGAGGGGSFGPRTKGKEGNGGGGSVTPKLQPDGLHGGGGSTVVPRDD PQFDNYVKICGGGGSVGTRPKDEVVRPKSRSSSRPATRGNSPAPKGGGGSCDNSPKGLLACQYNTGNFSDGFYPFTNSTLVREGGGGSTNVSNAQPNSGGVNTGGGGSMYGSYHPSCSFRPETINSGLWFGGGGSKSDGSRIQTRTEPGGGGSHNYNNIT (as SEQ ID NO.1 shown).

[0009] The nucleotide sequence of the gene encoding the recombinant fusion protein IBV:NS1 is as follows:

[0010] AAATTTGAAGGTTCTGGTGTTCCGGATAATGAAAACCTGAAAAATTCTCAGCAGCACGGTTATTGGCGTCGTCAGGCACGTTATAAACAGGGTAAAGGTGGTCGTAAACCGGTTCCGGATGCTTGGGGTGGTGGTGGTAGCGTTAAATCTCGTTCTAACCAGGGTACTCGTGATCCGGATAAATTCGACCAGTTCCCGCTGCGTTTCTCCGATGGCGGACCGGATGGCAACTTCCGCTGGGACTTCATCCCGCTGAACCGTGGTCGTTCTGGTCGTAGCACTGCGGCTGGCGGTGGTGGTAGCGCTGCGAGCAGCCGTGTTCCGAGCCGCGAAGGCAGCCGTGGTCGTCGTTCAGGTGCGGGCGGCGGCGGCTCCTTCGGCCCGCGTACCAAAGGCAAAGAAGGCAACGGCGGGGGCGGTTCCGTTACCCCGAAACTGCAGCCGGATGGTCTGCACGGTGGCGGCGGCAGCACCGTGGTTCCGCGCGATGATCCGCAGTTCGATAACTACGTTAAAATCTGCGGCGGTGGCGGCAGCGTTGGCACCAGGCCGAAAGATGAAGTTGTTCGTCCGAAATCTCGCAGCTCTAGCCGTCCGGCGACCCGCGGCAACAGCCCGGCGCCGAAAGGTGGTGGTGGTAGCTGTGACAACAGCCCGAAAGGGCTGCTGGCGTGCCAGTACAACACTGGCAACTTCAGCGATGGCTTCTACCCGTTCACCAACAGCACCCTGGTTCGTGAAGGCGGCGGCGGCAGCACCAACGTTAGCAACGCTCAGCCGAACAGCGGTGGCGTTAACACCGGTGGCGGCGGCAGTATGTACGGCAGCTACCACCCGAGCTGTAGCTTCCGTCCGGAAACCATTAACTCTGGTCTGTGGTTCGGTGGCGGTGGCTCTAAATCTGATGGTAGCCGTATCCAAACCCGTACCGAACCGGGTGGCGGCGGTAGCCATAACTATAACAACATCACC (as shown in SEQ ID NO.2).

[0011] A method for constructing the encoding gene of the recombinant protein described above includes the following steps:

[0012] Using restriction endonuclease ligation and overlap PCR ligation methods, the DNA encoding IBV:N, a multi-antigenic epitope tandem protein derived from the N protein of QX infectious bronchitis virus, and the DNA encoding IBV:S1, a multi-antigenic epitope tandem protein derived from the S1 protein of QX infectious bronchitis virus, were tandemly linked using a flexible linker to obtain the gene encoding the fusion recombinant protein IBV:NS1.

[0013] Furthermore, the encoding DNA of the multi-antigenic epitope tandem protein derived from the N protein of QX infectious bronchitis virus includes gene fragments of multiple antigenic epitopes tandemly with flexible linker spacers, and the codon-optimized encoding DNA IBV:N is chemically synthesized as shown in SEQ ID NO.2, 1-627bp, and the corresponding amino acid sequence is shown in SEQ ID NO.1, 1-209aa.

[0014] The DNA encoding the multi-antigenic epitope tandem protein derived from the S1 protein of QX infectious bronchitis virus comprises gene fragments of multiple antigenic epitopes tandemly with flexible linker spacers. The codon-optimized DNA encoding IBV:S1 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.

[0015] Furthermore, the restriction endonuclease ligation site ligation method involves introducing... Nco I, Eco RI Hind III. Xho I. Upstream and downstream primers for the DNA encoding the IBV:N and IBV:S1 multiantigenic epitope tandem proteins, containing four restriction endonuclease cleavage sites.

[0016] The Overlap PCR method introduces overlapping regions of DNA sequences into upstream and downstream primers encoding the DNA of a multi-antigenic epitope tandem protein, including:

[0017] The upstream primer of the DNA encoding the multi-antigenic epitope tandem protein of the N protein of QX infectious bronchitis virus (IBV:N) contains Nco I, Eco RI Hind III. Three restriction endonuclease sites, as shown in SEQ ID NO.3; the downstream primer contains a partially overlapping IBV:S1 sequence, as shown in SEQ ID NO.4;

[0018] The upstream primer encoding the multi-antigenic epitope tandem protein of the S1 protein of QX infectious bronchitis virus (IBV):S1 contains a partially overlapping IBV:N sequence, as shown in SEQ ID NO. 5; the downstream primer contains... Xho I. Restriction endonuclease site, as shown in SEQ ID NO.6.

[0019] A recombinant expression system for the recombinant protein described above, wherein the recombinant expression system includes a eukaryotic expression system or a prokaryotic expression system.

[0020] A recombinant expression plasmid expressing the recombinant protein described above, wherein the base vector of the recombinant expression plasmid includes pET-28a, and an upstream primer for the fusion recombinant protein encoding DNA IBV:NS1 is introduced. Nco I, Eco RI Hind III. Three restriction endonuclease sites, as shown in SEQ ID NO.3, with the downstream primer introducing the linker and... Xho I. Restriction endonuclease site, as shown in SEQ ID NO.6, is inserted into pET-28a via double digestion ligation. Nco I and Xho Between the I restriction enzyme sites, a recombinant expression plasmid pET28a-IBV:NS1 was constructed to fuse the recombinant protein IBV:NS1.

[0021] A method for inducing the expression of the recombinant protein described above includes the following steps: transforming the recombinant expression plasmid pET28a-IBV:NS1 into Escherichia coli. E. coli In Rosetta(DE3) competent cells, an E. coli genetically engineered expression strain, Ec-RD-pET28a-IBV:NS1, capable of expressing the fusion recombinant protein IBV:NS1, was obtained. The fusion recombinant protein IBV:NS1 was then obtained using IPTG-induced expression and inclusion body refolding methods.

[0022] The application of the recombinant protein described above or the recombinant protein obtained by the induction expression method described above in the preparation of a subunit vaccine for infectious bronchitis in chickens.

[0023] A subunit vaccine for infectious bronchitis in chickens, the subunit vaccine comprising an immune adjuvant and the recombinant protein described above or obtained by the induction expression method described above.

[0024] Furthermore, the immune adjuvant and recombinant protein are mixed at a volume ratio of 1:1, and the immune adjuvant is aluminum hydroxide sol adjuvant.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention selects multiple antigenic epitopes from the capsid protein of infectious bronchitis virus (IBV) to construct a fusion recombinant protein, IBV:NS1. The chicken IBV subunit vaccine prepared using this protein, due to the abundance of capsid protein antigenic epitopes, can stimulate the body to produce more types and quantities of protective antibodies, significantly improving its protective efficacy against chicken IBV. Simultaneously, the selection of multiple antigenic epitopes and the optimization of the codons encoding the IBV:NS1 DNA based on E. coli codon preference significantly increase the expression level of the IBV:NS1 fusion recombinant protein in the E. coli expression system, ensuring sufficient IBV:NS1 for vaccine preparation. In summary, the subunit vaccine prepared based on the IBV:NS1 fusion recombinant protein has advantages such as high antigen expression level, long antibody retention time, high purity, good safety, strong immunogenicity, and strong protective effect, effectively preventing chicken IBV. Attached Figure Description

[0027] Figure 1 This is a PCR amplification result diagram of the fusion recombinant protein encoding gene IBV:NS1 obtained by ligating the DNA XCIBV:N and XCIBV:S1, which are derived from two multi-antigen epitope tandem proteins, in step (2) of Example 1 of the present invention, using the Overlap PCR method; wherein: lane M is DL 5000 DNA Marker; lane 1 is the encoding gene IBV:NS1 of the fusion recombinant protein IBV:NS1.

[0028] Figure 2 The recombinant cloning plasmid pMD19-T-IBV:NS1 and the pET-28a vector containing the inserted fusion recombinant protein-coding gene in Example 2 of this invention were subjected to restriction endonuclease restriction enzyme assays. Nco I, Xho The image shows the agarose gel electrophoresis results of the double enzyme digestion products after identification. Lane M in lane I represents the DL 5000 DNA Marker, and lane M in lane II represents the DL 15000 DNA Marker. Lane 1 contains the IBV:NS1 fusion recombinant protein encoding gene after double enzyme digestion of the recombinant cloning plasmid pMD19-T-IBV:NS1. Lane 2 contains the undigested IBV:NS1 fusion recombinant protein encoding gene. Lane 3 contains the product of double enzyme digestion of the pET-28a vector. Nco I-pET28a- Xho Lane 4 contains the undigested pET-28a vector.

[0029] Figure 3This is a PCR amplification result of the encoding gene of the fusion recombinant protein IBV:NS1 obtained by double enzyme digestion and ligation of pMD19-TIBV:NS1 and pET-28a vector in Example 2 of the present invention; wherein: lane M is DL5000 DNA Marker; lane 1 is the encoding gene IBV:NS1 after double enzyme digestion and ligation.

[0030] Figure 4 This is an SDS-PAGE electrophoresis result of the recombinant protein IBV:NS1 fusion in step (1) of Example 3 of the present invention; wherein: lane M is a 10-250 kDa Marker; lane 1 is the total protein of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 without induction; lane 2 is the total protein of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 induced expression; lane 3 is the total protein of the supernatant of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 without induction; lane 4 is the total protein of the supernatant of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 induced expression; lane 5 is the total protein of the precipitated protein of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 induced expression; lane 6 is the total protein of the precipitated protein of the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 without induction.

[0031] Figure 5 This is an SDS-PAGE electrophoresis result of the purified recombinant fusion protein IBV:NS1 in step (2) of Example 3 of the present invention; wherein: lane M is a 10-180 kDa Marker; lane 1 is the pET28a empty vector; lane 2 is the total protein precipitated by the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1; lane 3 is the purified recombinant fusion protein IBV:NS1 induced by the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1.

[0032] Figure 6 This is a graph showing the change in serum antibody levels in chickens after protein immunization in step (2) of embodiment 4 of the present invention;

[0033] Figure 7 This is a survival curve of chickens after two immune challenges in the protective experiment of immune challenge in step (2) of embodiment 4 of the present invention. Detailed Implementation

[0034] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0035] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0036] Example 1: Construction of a cloning vector encoding the recombinant protein IBV:NS1 gene;

[0037] (1) Antigenicity prediction was performed on the amino acid sequences of two capsid proteins, N and S1, of the infectious bronchitis virus (IBV) QX genotype strain ck / CH / LJS / 101109 (GenBank accession number KX219794.1). Based on the characteristics of the epitopes, protein fragments located in the extracellular region with good hydrophilicity and concentrated epitopes were selected. Using the coding DNA nucleotide sequence of the protein flexible linker GGGGS, the coding DNA of the selected epitope-concentrated protein fragments was linked to construct two multi-epitaphedral tandem protein-coding DNAs, namely IBV:N and IBV:S1. The two multi-epitaphedral tandem protein-coding DNAs were directly synthesized chemically after codon optimization based on the codon usage preference of the E. coli expression system. DNA synthesis was completed by Shanghai Sangon Biotech Co., Ltd.

[0038] (2) Using the overlap PCR method, IBV:N and IBV:S1 were sequentially tandemly in the N-S1 order. Overlap primers for IBV:N and IBV:S1 were designed using SnapGene. Using the multi-antigenic epitope tandem protein-encoding DNA IBV:N and IBV:S1 as templates, the resulting DNA was amplified to obtain the IBV:N and IBV:S1-encoded multi-antigenic epitope tandem protein-encoding DNA. Nco I and Xho The IBV:N and IBV:S1 fusion recombinant proteins with different restriction enzyme sites at their 5' and 3' ends were amplified by PCR using the primers in Table 1 and the PCR amplification system and conditions in Table 2. The coding gene for IBV:NS1, a fusion recombinant protein with different restriction enzyme sites at both the 5' and 3' ends, was obtained. The specific steps are as follows:

[0039] Using the DNA IBV:N template encoding a multi-antigenic epitope tandem protein, and employing a template containing... Nco I, Eco RI Hind III. PCR amplification was performed using the upstream primer XCN:NS1-F (as shown in SEQ ID NO.3) containing three restriction endonuclease sites and the downstream primer XCN:NS1-R (as shown in SEQ ID NO.4) containing a partial homologous sequence of IBV:S1, yielding the DNA fragment XCIBV:N. Using the DNA encoding IBV:S1, a multi-antigenic epitope tandem protein, as a template, PCR amplification was performed using the upstream primer XCS1:NS1-F (as shown in SEQ ID NO.5) containing a partial homologous sequence of IBV:N and the downstream primer XCS1:NS1-R (as shown in SEQ ID NO.4). XhoPCR amplification was performed using the downstream primer XCS1:NS1-R (as shown in SEQ ID NO. 6) containing the restriction endonuclease site, amplifying the DNA fragment XCIBV:S1. Using XCIBV:N and XCIBV:S1 as templates, a method containing... Nco I, Eco RI Hind III. The upstream primer XCN:NS1-F (as shown in SEQ ID NO. 3) for the three restriction endonuclease sites and containing linker and Xho PCR amplification was performed using the downstream primer XCS1:NS1-R (as shown in SEQ ID NO. 6) at the restriction endonuclease site. The amplification product was a DNA fragment longer than 1000 bp. After the PCR reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and the results are shown below. Figure 1 As shown, there are clear and bright bands at 1000 bp and above. Afterwards, gel recovery was performed, and the recovered DNA fragments were ligated to the pMD-19T (Simple) vector using a T-vector ligation kit. The ligation system was incubated overnight at 16°C. The ligation system is shown in Table 3.

[0040] Table 1 Information on all primers

[0041]

[0042] Table 2 PCR amplification system and conditions

[0043]

[0044] Note: The PCR reaction program is 98℃ for 5 min; 35 cycles (98℃ for 15 s; 60℃ for 40 s; 72℃ for 30 s); 72℃ for 5 min; store at 4℃.

[0045] Table 3 Connection System

[0046]

[0047] (3) Transform the recombinant cloning plasmid containing the target DNA obtained after ligation in step (2) into competent E. coli cells. E. coli Positive transformants from DH5α were selected and sent to Sangon Biotech Co., Ltd. for sequencing.

[0048] Example 2: Construction of a prokaryotic gene-engineered bacterium fused with recombinant protein IBV:NS1;

[0049] The recombinant cloning plasmid pMD19-T-IBV:NS1, containing the inserted target DNA fragment, was extracted using a plasmid extraction kit. The coding gene for the fusion recombinant protein was constructed using a restriction endonuclease ligation method. The specific steps are as follows:

[0050] Recombinant cloning plasmid pMD19-T-IBV:NS1 is used Nco I, Xho I was subjected to double enzyme digestion, and the system is shown in Table 4; pET-28a vector was used... Nco I, Xho I was subjected to double enzyme digestion, and the system is shown in Table 5. The system was incubated at 37℃ for 1 h for digestion. After digestion, the digestion products were subjected to 1% agarose gel electrophoresis, and the results are shown below. Figure 2 As shown in Table 6, the recombinant cloning plasmid pMD19-T-IBV:NS1 showed two bands after double digestion, consistent with the expected size, with no nonspecific bands; the pET-28a vector showed one band after double digestion, consistent with the expected size, with no nonspecific bands. The target DNA fragments were recovered using a DNA gel extraction kit, yielding fragments with sticky ends at different restriction enzyme sites. These fragments were then ligated using T4 DNA ligase to obtain the encoding gene of the fusion recombinant protein. The ligation system (as shown in Table 6) was incubated overnight in a 16°C water bath. The ligation product was then transformed into *E. coli*. E. coli In Rosetta(DE3), positive clones successfully transformed with the recombinant expression plasmid were identified by PCR. The PCR amplification results of the gene encoding the recombinant protein IBV:NS1 were as follows: Figure 3 As shown, a clear and bright single band is present at a position greater than 1000 bp. The selected positive clones were sent to a biotechnology company for sequencing. The plasmid with correct sequencing results is the prokaryotic expression plasmid pET28a-IBV:NS1 used to express the recombinant fusion protein IBV:NS1. [The text then abruptly shifts to a different topic:] ...containing this recombinant expression plasmid... E. coli The Rosetta(DE3) expression strain was named Ec-RD-pET28a-IBV:NS1.

[0051] Table 4. Recombinant cloning plasmid pMD19-T-IBV:NS1 double enzyme digestion system

[0052]

[0053] Table 5 pET-28a vector double enzyme digestion system

[0054]

[0055] Table 6 Target Fragment Connectivity System

[0056]

[0057] Example 3: Expression and purification of the fusion recombinant protein IBV:NS1;

[0058] (1) The genetically engineered expression strain Ec-RD-pET28a-IBV:NS1 was inoculated into 5 mL of liquid LB medium containing 100 μg / mL kanamycin and cultured at 37℃ for 2 h. Then, IPTG was added to bring the final concentration to 0.5 mmol / L, and the culture was induced for 8 h. The successful expression of the target fusion recombinant protein IBV:NS1 was verified by SDS-PAGE electrophoresis. The induced expression strain was sonicated and the sonicated bacterial solution was centrifuged at 8000 rpm and 4℃ for 10 min. The supernatant and precipitate were collected, and the expression level and soluble state of the target protein IBV:NS1 in the genetically engineered expression strain were detected by SDS-PAGE electrophoresis.

[0059] The SDS-PAGE electrophoresis results of the genetically engineered expression strain Ec-RD-pET28a-IBV:NS1 are as follows: Figure 4 As shown in the figure, the fusion recombinant protein IBV:NS1 can be successfully induced to express, and the total protein precipitated by the genetically engineered expression bacterium Ec-RD-pET28a-IBV:NS1 is good, indicating that the main expression form of the protein is inclusion body expression.

[0060] (2) Refolding of the fusion recombinant protein IBV:NS1;

[0061] After the genetically engineered expression strain Ec-RD-pET28a-IBV:NS1 was induced to express its gene using IPTG, the bacterial cell pellet was collected. Given...

[0062] SDS-PAGE analysis showed that the recombinant protein was expressed in inclusion bodies, so inclusion body refolding purification was used to purify the recombinant protein. The specific procedures are as follows:

[0063] ① Expanded culture and induced expression: 5 mL of Ec-RD-pET28a-IBV:NS1 bacterial culture that had been cultured overnight at 37℃ was inoculated into 1 L of sterile LB medium, 100 μg / mL kanapenem was added, and the culture was incubated at 37℃ and 180 rpm for 2 h. Then IPTG was added to make the final concentration 0.5 mmol / L, and the culture was induced at 37℃ and 180 rpm for another 8 h. After induction, the bacterial culture was centrifuged at 8000 rpm and 4℃ for 10 min, and the bacterial pellet was collected.

[0064] ② Bacterial cell washing and inclusion body collection: The collected bacterial cells were washed twice with 1×PBS, centrifuged at 8000 rpm and 4℃ for 10 min, and finally the bacterial cell precipitate was collected.

[0065] The bacterial cells were suspended in TE buffer at a ratio of 1:100 (wet weight of bacterial cells to TE buffer (TE buffer formulation: Tris 2.4228g, EDTA 0.2923g, brought to 1L, pH adjusted to 8.5) and then sonicated (φ6, 10% power, sonication for 3s, pause for 5s, for a total of 40min). After disruption, the cells were centrifuged at 8000rpm and 4℃ for 40min and inclusion bodies were collected.

[0066] ③Inclusion body washing:

[0067] Add washing buffer I at a ratio of 1:50 (g / mL) to the wet weight of inclusion bodies (washing buffer I formulation: Tris 2.4228g, EDTA 0.2923g, 10mL Tritonx-100, adjusted to 1L). Wash at 200rpm for 2h at room temperature, then centrifuge at 8000rpm and 4℃ for 40min to collect the inclusion body precipitate. Repeat this washing step once.

[0068] The collected inclusion body precipitate was added to Wash Buffer II at a ratio of 1:50 (g / mL) (Wash Buffer II formulation: Tris 2.4228g, EDTA 0.2923g, 10mL Tritonx-100, 120.12g Urea, adjusted to 1L, pH 8.5). The mixture was washed at 200rpm for 2 hours at room temperature, then centrifuged at 8000rpm and 4℃ for 40 minutes to collect the inclusion body precipitate. This washing step was repeated once.

[0069] ④ Inclusion body denaturation: Dissolve the washed inclusion bodies in denaturation buffer (denaturation buffer formula: Tris 2.4228g, EDTA 2.923g, Urea 480.48g, bring to a final volume of 1L, adjust pH to 9.5) at a ratio of 1:15, stir overnight at 4°C and 120rpm, and then centrifuge at 8000rpm and 4°C for 40min. The supernatant obtained is the denatured inclusion body.

[0070] ⑤ Denatured protein refolding: Pour the denatured protein into a treated dialysis bag. Using a denaturing buffer to refolding buffer volume ratio of 1:100, immerse the dialysis bag containing the denatured protein sequentially in refolding buffer I (Tris 2.4228g, GSH 0.61464g, GSSG 0.122526g, Urea 360.36g, adjusted to 1L, pH 9.5), refolding buffer II (Tris 2.4228g, GSH 0.61464g, GSSG 0.122526g, Urea 240.24g, adjusted to 1L, pH 9.5), and refolding buffer III (Tris 2.4228g, GSH 0.61464g, GSSG 0.122526g, Urea 240.24g, adjusted to 1L, pH 9.5), and Urea 3 (Tris 2.4228g, GSH 0.61464g, GSSG 0.122526g, Urea 360.36g, adjusted to 1L, pH 9.5). The denaturing solution was prepared by refolding with 120.12g of Tris (adjusted to 1L and pH 9.5) and refolding buffer IV (Tris 2.4228g, GSH 0.61464g, GSSG 0.122526g, adjusted to 1L and pH 9.5). The refolding system was placed in a dialysis cabinet at 4℃ with very slow stirring, and each dialysis session lasted 6-12 hours. Finally, the denaturing solution was dialyzed into PBS buffer at pH 7.4, centrifuged at 8000 rpm at 4℃ for 30 min, and the supernatant was collected and filtered through a 0.22nm filter for sterilization.

[0071] ⑥ Protein Concentration Assay and Detection: The concentration of the purified target protein was determined using the BCA protein concentration assay kit from Beyotime Biotechnology Co., Ltd. The concentration of the purified recombinant fusion protein IBV:NS1 was determined to be 1 mg / mL. The SDS-PAGE electrophoresis results of the purified recombinant fusion protein IBV:NS1 are shown below. Figure 5 As shown, the purified fused recombinant protein IBV:NS1 exhibits the target protein band at the expected size.

[0072] Example 4: Preparation and immunoprotective analysis of genetically engineered subunit vaccine for infectious bronchitis in chickens;

[0073] (1) Preparation of subunit vaccines;

[0074] The fusion recombinant protein IBV:NS1 purified in Example 3 was diluted to 100 μg / mL and mixed with aluminum hydroxide sol adjuvant at a volume ratio of 1:1 to prepare a subunit vaccine.

[0075] (2) Immunogenicity analysis of subunit vaccines;

[0076] ① Immunogenicity analysis;

[0077] Fourteen-day-old specific pathogen-free SPF Hy-Line White chickens were selected and divided into two groups of six each. One group was the immunization group, immunized with a subunit vaccine prepared from the recombinant protein IBV:NS1 fusion protein; the other group was the PBS control group, injected with the same volume of PBS containing aluminum hydroxide sol as adjuvant. The immunization cycle was 14 days, and the immunization program consisted of two immunizations administered subcutaneously in the neck. Starting one week after the initial immunization, serum samples were collected weekly from the subwing vein for antibody titer monitoring. The antibody titer in the serum was detected using an indirect ELISA method, and the p / n ratio was calculated. The results are as follows: Figure 6 As shown in the results, the subunit vaccine prepared from the fusion recombinant protein IBV:NS1 can stimulate the body to produce antibodies, and the antibodies are maintained for a long time. This indicates that the prepared fusion recombinant protein IBV:NS1 has good immunogenicity, and the vaccine composed of it and adjuvant can stimulate the body to produce antibodies with strong binding ability and long duration.

[0078] ② Animal challenge and immune protection experiments;

[0079] Grouping and immunization of experimental chickens;

[0080] Fourteen-day-old SPF Hy-Line White laying hens were selected, with six hens in each group. The immunization cycle was 14 days, and the immunization program consisted of two immunizations administered via subcutaneous injection in the neck. Specific grouping details are as follows:

[0081] Immunization experimental group: The two-immunization group was immunized with the above-prepared subunit vaccine (100 μg protein / bird) at 14 days of age, and challenged 14 days after immunization;

[0082] Healthy control group: group without immunization and without virus challenge;

[0083] Challenge control group: The challenge group that received PBS injection.

[0084] Protective testing against viral infection;

[0085] Following the second immunization, chickens in both the immunized experimental group and the challenge control group were subcutaneously injected into the neck with the M41 isolate at 100 TCID50. 50 The chickens were challenged with a specific dosage. For one week after challenge, the clinical symptoms of the chickens were observed daily, and mortality was recorded. Necropsys were performed on the dead chickens, and lesions were observed. The results of the protective effect experiment were as follows: Figure 7 As shown, none of the chickens in the immunized experimental group developed the disease, with a mortality rate of 0% after challenge and a protection rate of 100%. All chickens in the challenge control group developed the disease and died after challenge. No chickens in the healthy control group developed the disease or died. These results indicate that the subunit vaccine prepared from the recombinant protein IBV:NS1 provides complete protection, with two immunizations providing 100% protection.

[0086] Conclusion: The fusion recombinant protein IBV:NS1 of this invention exhibits good immunogenicity, and its subunit vaccine, composed of IBV:NS1 and adjuvant, can stimulate the body to produce antibodies with strong binding capacity and long duration of action. The subunit vaccine prepared from the fusion recombinant protein IBV:NS1 of this invention requires only two immunizations to achieve complete protection, significantly reducing immunization costs. This demonstrates that the subunit vaccine prepared from the fusion recombinant protein IBV:NS1 is both highly effective and economical.

[0087] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

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

2.

2. A method for constructing the encoding gene of the recombinant protein according to claim 1, characterized in that, Includes the following steps: Using restriction endonuclease ligation and overlap PCR ligation methods, the DNA encoding IBV:N, a multi-antigenic epitope tandem protein derived from the N protein of QX infectious bronchitis virus, and the DNA encoding IBV:S1, a multi-antigenic epitope tandem protein derived from the S1 protein of QX infectious bronchitis virus, were tandemly linked using a flexible linker to obtain the gene encoding the fusion recombinant protein IBV:NS1.

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

4. The construction method according to claim 2, characterized in that, The restriction endonuclease cleavage site ligation method is to introduce... Nco I, Eco RI Hind III. Xho I. Upstream and downstream primers for the DNA encoding the IBV:N and IBV:S1 multiantigenic epitope tandem proteins, containing four restriction endonuclease cleavage sites. The Overlap PCR method introduces overlapping regions of DNA sequences into upstream and downstream primers encoding the DNA of a multi-antigenic epitope tandem protein, including: The upstream primer of the DNA encoding the multi-antigenic epitope tandem protein of the N protein of QX infectious bronchitis virus (IBV:N) contains Nco I, Eco RI Hind III. Three restriction endonuclease sites, as shown in SEQ ID NO.3; the downstream primer contains a partially overlapping IBV:S1 sequence, as shown in SEQ ID NO.4; The upstream primer encoding the multi-antigenic epitope tandem protein of the S1 protein of QX infectious bronchitis virus (IBV):S1 contains a partially overlapping IBV:N sequence, as shown in SEQ ID NO. 5; the downstream primer contains... Xho I. Restriction endonuclease site, as shown in SEQ ID NO.

6.

5. A recombinant expression system for the 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 expressing the recombinant protein of claim 1, characterized in that, The recombinant expression plasmid is based on the pET-28a vector, and the upstream primer of the fusion recombinant protein encoding DNA IBV:NS1 is introduced into it. Nco I, Eco RI Hind III. Three restriction endonuclease sites, as shown in SEQ ID NO.3, with the downstream primer introducing the linker and... Xho I. Restriction endonuclease site, as shown in SEQ ID NO.6, is inserted into pET-28a via double digestion ligation. Nco I and Xho Between the I restriction enzyme sites, a recombinant expression plasmid pET28a-IBV:NS1 was constructed to fuse the recombinant protein IBV:NS1.

7. A method for inducing the expression of the recombinant protein according to claim 1, characterized in that, Includes the following steps: The recombinant expression plasmid pET28a-IBV:NS1 was transformed into E. coli. E. coli In Rosetta(DE3) competent cells, an E. coli genetically engineered expression strain, Ec-RD-pET28a-IBV:NS1, capable of expressing the fusion recombinant protein IBV:NS1, was obtained. The fusion recombinant protein IBV:NS1 was then obtained using IPTG-induced expression and inclusion body refolding methods.

8. The use of a recombinant protein according to claim 1 or a recombinant protein obtained by the induction expression method according to claim 7 in the preparation of a subunit vaccine for infectious bronchitis in chickens.

9. A subunit vaccine for infectious bronchitis in chickens, characterized in that, The subunit vaccine comprises an immune adjuvant and the recombinant protein of claim 1 or the recombinant protein obtained by the induction expression method of claim 7.

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

Images