Optimized recombinant chicken double-cytokine coding gene as well as preparation method and application of corresponding protein

Abstract

The invention discloses an optimized recombinant chicken double-cell factor as well as a preparation method and application thereof. The chicken recombinant double-cell factor coding gene provided by the invention contains complete ChIL-2 and ChIFN-alpha structures. The preparation process comprises the following steps: selecting a pichia pastoris expression system; optimizing the recombinant chicken double-cell factor according to the codon preference of pichia pastoris; inserting the optimized gene sequence into an expression vector, and introducing host bacteria to construct a recombinant yeast engineering strain; carrying out inoculated culture by using the obtained recombinant yeast, and screening strains containing target protein based on expression level to obtain required culture bacteria; and breeding and culturing the obtained high-expression-level culture bacteria. According to the invention, ChIL-2 and ChIFN-alpha are connected through a modified flexible linker, and a nucleotide sequence is replaced by preferred codons of pichia pastoris, so that a chicken recombinant cell factor can be secreted to a supernatant, the defect of ChIL-2 expression quantity is overcome, and the problem that proteins of ChIL-2 and ChIFN-alpha mainly exist in bacteria and inclusion bodies in prokaryotic expression is solved.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering biopharmaceuticals, and relates to the optimization of the coding gene of chicken double cytokine, and a method and application of preparing chicken double cytokine by using a eukaryotic expression system. Background Art

[0002] One of the major challenges facing my country's livestock industry is production losses caused by pathogenic microorganisms. With the ban on antibiotics in animal feed, natural agents with broad-spectrum antiviral activity, such as cytokines, have become a research hotspot to mitigate these negative impacts. Chicken interferon-α (ChIFN-α) is a multifunctional cytokine with biological functions, including antiviral and antimicrobial properties, anti-tumor activities, and immune regulation. When ChIFN-α is applied to tissue cells, it stimulates the body to gain corresponding functions, making it a green, highly effective, and multifunctional immunotherapeutic and immunopotentiator. For example, when ChIFN-α is injected intramuscularly into 42-day-old SPF chickens infected with Newcastle disease virus for 8 days, it reduces mortality and increases survival rates to 25%-48%. Furthermore, treatment of chicken fibroblasts with ChIFN-α can reduce Rous sarcoma virus titers by 25-fold.

[0003] Interleukin-2 (ChIL-2) plays a crucial role in the growth and proliferation of many immune cells. It maintains the long-term growth of T lymphocytes in vitro, promotes NK cell proliferation, induces IFN production, and clears intracellular pathogens. It can enhance cellular immunity and boost vaccine antibodies, and is therefore frequently studied as an immunotherapeutic and immunopotentiator. For example, studies have shown that IL-2 can significantly reduce the recurrence rate of herpes simplex virus in guinea pigs, and that ChIL-2 injection into chicken embryos can reduce Salmonella infection. Recombinant human IL-2 can enhance the immune response of live Salmonella typhi vaccine and capsular polysaccharide subunit vaccine. When recombinant bovine IL-2 is used in combination with live herpesvirus type 1 vaccine, serum neutralizing antibody titers increase sixfold compared to the vaccine alone.

[0004] However, although ChIFN-α and ChIL-2 have good antiviral therapeutic effects and immune-enhancing effects, there is currently no chicken dual cytokine product on the market that contains both ChIFN-α and ChIL-2 fusion proteins. It has not obtained a new veterinary drug certificate in my country, and no imported recombinant ChIFN-α has obtained an import registration certificate. This is because the current expression of chicken recombinant dual cytokines mainly uses a prokaryotic expression system (Escherichia coli). Studies have shown that although E. coli has the advantages of fast growth and reproduction and simple cultivation, the target protein expressed in E. coli cannot be correctly folded and modified after expression. The target protein is mainly present in the cell and rarely secreted outside the cell. In order to increase the extracellular expression of chicken recombinant dual cytokines, some studies have attempted to remove the signal peptide sequences of IFN-α and IL-2. However, after removing the signal peptide sequence, the target protein is still mainly present in the cell or inclusion body, and may cause changes in the tertiary structure of the target protein, thereby affecting the related functions of the target protein. More importantly, in order to obtain the target protein in Escherichia coli, it is necessary to collect and lyse the bacteria, collect the supernatant, remove the bacterial endotoxins, and purify and renature the target protein. These operations involve many steps, and the temperature and reagents must be strictly controlled, resulting in a large loss of target protein. When used in the production of livestock and poultry biological products in the future, the product preparation process is difficult and costly, which is not conducive to market promotion and sales. Summary of the Invention

[0005] Purpose of the Invention

[0006] To address the shortcomings of the existing technology, the present invention proposes a recombinant chicken dual cytokine containing a complete ChIL-2 and ChIFN-α structure. ChIL-2 and ChIFN-α are connected by a modified flexible linker, and the nucleotide sequence is replaced with the preferred codons of Pichia pastoris, enabling the secretion of the chicken recombinant cytokine into the supernatant. This overcomes the shortcomings of ChIL-2 expression and the problem that ChIL-2 and ChIFN-α proteins are primarily present in bacteria and inclusion bodies during prokaryotic expression. Furthermore, the present invention provides a strain capable of efficiently expressing the chicken recombinant cytokine, which can significantly improve the production efficiency of the chicken recombinant cytokine.

[0007] Principle Description

[0008] The Pichia pastoris expression system is a eukaryotic expression system that combines the advantages of rapid growth and simple culture of prokaryotes with the complete membrane system of eukaryotes. However, direct use of the Pichia pastoris system for the preparation of dual cytokines has limited yield. The present invention optimizes the Pichia pastoris system.

[0009] This invention connects ChIFN-α and ChIL-2 via a flexible linker to develop a chicken dual cytokine product. Using an optimized Pichia pastoris expression system, the recombinant chicken dual cytokine was optimized according to Pichia pastoris codon preference. After multiple screening procedures, including combined MM and MD screening and screening with different concentrations of G418, a yeast strain containing the target protein was obtained that is fast-growing and easy to culture. This allows the target protein to be correctly folded, modified, and secreted outside the cell. Without the need for endotoxin removal, simple physical operations such as concentration and ultrafiltration yield a target protein with a purity exceeding 90%. This protein maintains high antiviral and immunopotentiator activity, helping animals resist viral infection and boosting antibody levels after vaccination.

[0010] The product of the present invention contains a ChIFN-α and ChIL-2 fusion protein. When expressed in Pichia pastoris, the fusion protein is secreted into the supernatant. The supernatant is collected and subjected to 10KD and 50KD ultrafiltration to obtain the target protein with a purity of over 90%. The recombinant chicken dual cytokine of the present invention is expressed in a soluble form, has a high expression level, requires few and simple purification steps, and is low-cost, suitable for later industrial production and conducive to market promotion and sales.

[0011] The optimized Pichia pastoris expression system has high yield, simple product purification, simple fermentation process, and greatly reduces biological costs, which is something that other prokaryotic and eukaryotic expression systems cannot achieve. It is suitable for the industrial production and preparation of recombinant proteins.

[0012] Specifically, the present invention provides a chicken recombinant dual cytokine encoding gene, characterized in that the chicken recombinant dual cytokine expression gene contains complete ChIL-2 and ChIFN-α structures.

[0013] Preferably, the chicken recombinant dual cytokine encoding gene comprises the nucleotide sequences in SEQ ID No. 1 and SEQ ID No. 2 or the nucleotide sequences in SEQ ID No. 3 and SEQ ID No. 4 in the sequence listing.

[0014] On the other hand, the present invention provides a method for preparing the chicken recombinant double cytokine fusion protein, characterized in that the method comprises:

[0015] Step S1, selecting a Pichia pastoris expression system;

[0016] Step S2, connecting the ChIL-2 and ChIFN-α sequences to construct a recombinant chicken dual cytokine, and optimizing the sequence according to the codon preference of Pichia pastoris to obtain the gene sequence ChIL-2-IFN-α that fused and expressed ChIL-2 and ChIFN-α;

[0017] Step S3, connecting the gene sequence ChIL-2-IFN-α to the target vector to construct a recombinant vector, and transferring the constructed recombinant vector into competent cells of the target strain to obtain recombinant yeast;

[0018] Step S4: using the obtained recombinant yeast to inoculate and culture, and detecting the expression level of the chicken recombinant dual cytokine in the recombinant yeast, and screening strains containing the target protein based on the expression level to obtain the desired cultured bacteria with high expression level;

[0019] Step S5, multiplying and culturing the obtained cultured bacteria with high expression levels;

[0020] Step S6: inoculating the cultured bacteria with high expression levels into the culture medium and inducing the cultured bacteria to produce proteins containing the recombinant chicken double cytokine.

[0021] Preferably, the recombinant yeast comprises the recombinant yeast with the deposit number of CCTCC M 20232731, deposited at China Center for Type Culture Collection, Wuhan University, China, on December 29, 2023, and named Pichia pastoris SDLCUSJY1.

[0022] Preferably, the primers used to amplify ChIL-2 and ChIFN-α sequences include:

[0023] ChIL-2-F:5'-ATGATGTGCAAAGTACTGATCTTT-3'

[0024] ChIL-2-R:5'-TTATTTTTTGCAGATATCTCACAAA-3'

[0025] ChIFN-α-F:5'-ATGGCTGGCCTGCAAGCCCACAG-3'

[0026] ChIFN-α-R:5'-CTAAGTGCGCGTGTGCCTTGTGAG-3'

[0027] Preferably, in step S3, a flexible linker is used to connect ChIL-2 and ChIFN-α, and the sequence of the flexible linker is shown as SEQ ID No. 6 in the sequence listing.

[0028] On the other hand, the present invention provides a use of Pichia pastoris for preparing chicken recombinant dual-cytokine. Preferably, the Pichia pastoris is a yeast with a preservation number of CCTCC M 20232731.

[0029] On the other hand, the present invention provides an application of the chicken recombinant double cytokine, wherein the chicken recombinant double cytokine is used for inoculation into poultry to thereby inhibit virus replication in the poultry.

[0030] Preferably, the chicken recombinant double cytokine is used for inoculation into chickens.

[0031] Preferably, the chicken recombinant double cytokine is used for inoculation into chicken embryos.

[0032] Technical Effects

[0033] 1. The present invention adopts a yeast expression system to prepare dual cytokines, thus avoiding the problems of inclusion bodies and incorrect protein folding in prokaryotic expression.

[0034] 2. The present invention obtains an engineered bacterium that efficiently produces chicken recombinant double cytokines. The use of this engineered bacterium can greatly increase the cultivation rate of the double cytokines.

[0035] 3. The chicken recombinant double cytokine purification process of the present invention is simple and low in cost.

[0036] 4. The product of the present invention has the effect of resisting chicken viral diseases.

[0037] 5. The product of the present invention can be used as an immune adjuvant to improve the effect of vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The figure shows the enzyme digestion results obtained in the examples of the present invention, wherein M: 10000bp DNA Marker; 1: pPIC9K empty vector double enzyme digestion product; 2: recombinant plasmid ChIL2-IFNα / pPIC9K double enzyme digestion product; 3: recombinant plasmid ChIL2-IFNα / pPIC9K.

[0039] Figure 2 The PCR results of the plasmids in the examples of the present invention are shown, where M: 2000 bp DNA Marker; 1: positive control; 2: recombinant plasmid amplified with target gene primers; 3: recombinant plasmid amplified with vector universal primers; 4: pPIC9K empty vector amplified with vector universal primers; 5: pPIC9K empty vector amplified with target gene primers.

[0040] Figure 3 The PCR results of recombinant yeast in the examples of the present invention are shown, wherein M: 2000 bp DNA Marker; 1: positive control; 2-12: recombinant yeast colonies; 13: negative control.

[0041] Figure 4The SDS-PAGE identification results of the recombinant ChIL2-IFNα protein in the example of the present invention, wherein M: 10-180kD protein marker; 1: expression product of the pPIC9K empty vector strain; 2-7: expression product of the ChIL2-IFNα / pPIC9K recombinant bacteria.

[0042] Figure 5 The expression results of recombinant ChIL2-IFNα induced by different concentrations of methanol in the examples of the present invention, where M: 10-180kD protein marker; 1: expression product of the pPIC9K empty vector strain transformed; 2-7: expression product induced by 0.5%, 1%, 2%, 3%, 4%, and 5% methanol.

[0043] Figure 6 These are the expression results of recombinant ChIL2-IFNα at different induction times in the examples of the present invention, where M: 10-180 kD protein marker; 1: expression product of the pPIC9K empty vector strain transformed; 2-8: expression product induced at 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, and 144 h.

[0044] Figure 7 These are the expression results of recombinant ChIL2-IFNα under different induction temperature conditions in the examples of the present invention, where M: 10-180kD protein marker; 1: expression product of the pPIC9K empty vector strain transformed; 2-5: expression product induced at 26°C, 28°C, 30°C, and 32°C.

[0045] Figure 8 These are proteins purified by different methods in the examples of the present invention, where M: Marker; 1: sample treated with Ni column; 2: sample treated with ultrafiltration tube.

[0046] Figure 9 The results of the verification of the inhibitory effect of ChIL2-IFNα protein on VSV in the examples of the present invention are shown, wherein AC: CEF cells infected with VSV after treatment with doubly diluted recombinant ChIL2-IFNα; D: normal CEF cells not infected with VSV; E: CEF cells of the VSV virus control group.

[0047] Figure 10 This is the result of the antiviral effect of the chicken recombinant double cytokine in the embodiment of the present invention.

[0048] A: Cytokines against influenza virus; B: Cytokines against Newcastle disease virus.

[0049] Figure 11This is a diagram showing the effect of chicken recombinant double cytokine as a vaccine adjuvant in an embodiment of the present invention, wherein A: antibody titer after immunization with avian influenza vaccine; B: antibody titer after immunization with Newcastle disease vaccine. DETAILED DESCRIPTION

[0050] The present invention will be described in detail below with reference to the accompanying drawings and embodiments thereof, but the protection scope of the present invention is not limited to the scope of the embodiments described herein.

[0051] Example 1 Optimization of chicken recombinant dual cytokine expression genes

[0052] To address the lack of chicken dual cytokine products on the market, the present invention uses Primer premier 5 software to design primers based on the ChIL-2 and ChIFN-α gene sequence numbers (HQ008781 and DQ906157) provided in GenBank. The amplification primers for ChIL-2 and ChIFN-α are designed as follows:

[0053] ChIL-2-F:5'-ATGATGTGCAAAGTACTGATCTTT-3'

[0054] ChIL-2-R:5'-TTATTTTTTGCAGATATCTCACAAA-3'

[0055] ChIFN-α-F:5'-ATGGCTGGCCTGCAAGCCCACAG-3'

[0056] ChIFN-α-R:5'-CTAAGTGCGCGTGTGCCTTGTGAG-3'

[0057] Choose Roman brown-shell laying hens with a higher market share, and use 100EID 50 After infecting this breed of chicken with H9 subtype influenza virus, spleen lymphocytes were aseptically isolated and stimulated with Con A. RNA was extracted using an RNA extraction kit (Cat. No. RC311-01, Nanjing Novozymes Biotech Co., Ltd.) according to the instructions. After reverse transcription into cDNA, the genes ChIFN-α and ChIL-2 were amplified. The reaction system and procedures are shown in Tables 1 and 2:

[0058] Table 1 PCR reaction system

[0059]

[0060] Table 2 PCR reaction procedure

[0061]

[0062] The PCR products were subjected to gel electrophoresis, excised, and recovered using a gel DNA extraction kit (Cat. No. DC301-01, Nanjing Novozymes Biotech Co., Ltd.) according to the manufacturer's instructions. The recovered PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, yielding the following sequencing results (SEQ ID No. 1 and SEQ ID No. 2):

[0063] ChIL-2:

[0064]

[0065] ChIFN-α:

[0066]

[0067] According to the above sequencing results, flexible linker

[0068] (5'-GGAGGAGGAGGTTCAGGAGGAGGAGGTTCAGG

[0069] AGGAGGAGGTTCT-3') was used to connect ChIL-2 and ChIFN-α, and the restriction site EcoRI was added to the N-terminus of ChIL-2, the restriction site NotI was added to the C-terminus of ChIFN-α, and a 6×His tag was added to the C-terminus of the restriction site NotI. The gene sequence was codon-optimized according to yeast codon preference to obtain the gene sequence expressing ChIL-2 and ChIFN-α fusion, named ChIL-2-IFN-α (SEQ ID No. 5). It was sent to Suzhou Hongxun Biotechnology Co., Ltd. for artificial synthesis, and the sequence results are as follows:

[0070] IL-2-IFN-a:

[0071]

[0072] The optimized ChIL-2 sequence is shown as SEQ ID No. 3 in the sequence listing, and the optimized ChIFN-α sequence is shown as SEQ ID No. 4 in the sequence listing, which are identical to the following sequences:

[0073] ChIL-2-optimized sequence

[0074]

[0075] ChIFNα-optimized sequence

[0076]

[0077] Example 2 Construction of recombinant yeast engineering strains

[0078] The ChIL2-IFNα gene and pPIC9K vector were double-digested with EcoRI and NotI restriction endonucleases, respectively. The double-digestion procedure is shown in Table 3. The digestion reaction system was placed on a floating plate and incubated in a 37°C water bath for 30 minutes. The double-digested ChIL2-IFNα gene and pPIC9K vector were then recovered using a DNA product purification kit (Cat. No. DP204-02, Tiangen Biochemical Technology Co., Ltd.). The ChIL2-IFNα gene and pPIC9K vector were ligated using T4 DNA ligase, incubated in a 16°C water bath overnight, and inactivated at 65°C for 10 minutes. The ligation procedure is shown in Table 4.

[0079] Table 3 Enzyme digestion system

[0080]

[0081] Table 4 Connection system

[0082]

[0083]

[0084] Take 5 μL of the ligation product and add it to DH5α competent cells (Cat. No.: D1031S, Biyuntian Biotechnology Co., Ltd.) and transform according to the instructions. Spread the transformed E. coli on LB plates (containing Amp 50 μg / mL), culture at 37°C for 14-16 hours, pick out white single colonies, place them in LB liquid culture medium (containing Amp 50 μg / mL), and culture on a shaker at 37°C and 180 r / min. After 16 hours of shaking culture, take the bacterial solution and extract the plasmid according to the instructions (Cat. No.: DP103-02, Tiangen Biochemical Technology Co., Ltd.). The extracted plasmid was double-digested with EcoR I and Not I, and the enzyme digestion effect was identified by gel electrophoresis, as shown in the following figure. Figure 1 As shown, the results show that the ChIL2-IFNα gene was successfully connected to the pPIC9K vector, and the target band was cut out under the double enzyme digestion of EcoR I and Not I. The recombinant plasmid was verified by PCR, and PCR amplification was performed using AOX1 universal primer and ChIL-2, ChIFN-α upstream and downstream primers (see Example 1). The amplification method is as shown in Tables 1 and 2 of Example 1. The electrophoresis results are shown in Figure 2 As shown, the results showed that the target band of the correct size was obtained after PCR, indicating that the ChIL2-IFNα gene was successfully linked to the pPIC9K vector. The recombinant vector was named pPIC9K-ChIL2-IFNα. The target band was recovered from the gel and sent to Qingdao Ruibo for sequencing, and the sequencing results were correct.

[0085] The recombinant plasmid pPIC9K-ChIL2-IFNα was linearized using a single enzyme digestion with Sac I. The reaction system is shown in Table 6. The linearized plasmid was then recovered using a DNA product purification kit. The linearized pPIC9K-ChIL2-IFNα was added to Pichia pastoris competent cells for transformation. The transformed bacterial solution was plated onto MD plates and inverted for 3-5 days. After colonies grew, single colonies were picked from the MD plates and inoculated onto MM plates for phenotypic identification. The inverted plates were then incubated for 2-3 days. Once colonies grew, they were inoculated onto YPD plates containing 0.5, 1.0, 1.5, 2.0, 3.0, or 4.0 mg / mL G418. The cells were inverted at 30°C for 2-3 days. Single colonies were then picked and incubated in 3 mL of YPD liquid medium for 14-16 hours. Yeast was harvested by centrifugation, and yeast DNA was extracted according to the instructions of the Yeast DNA Extraction Kit (Cat. No. 78870, Thermo Scientific Pierce). PCR identification was performed (the protocol described above). Identification results such as Figure 3 As shown, the results showed that the target band of the correct size was obtained after PCR, indicating that the ChIL2-IFNα gene was successfully integrated into the yeast genome. The recombinant yeast was named GS115 / pPIC9K-ChIL2-IFNα. The target bands recovered from the gel were selected from 2, 3, 5, 6, 8, and 11 recombinant yeasts and sent to Qingdao Ruibo Company for sequencing. The sequencing results were correct.

[0086] Table 6 Enzyme digestion system

[0087]

[0088]

[0089] Example 3 Expression and purification of chicken recombinant dual cytokines

[0090] 1. Screening of superior recombinant yeast.

[0091] Protein expression and pretreatment. Four recombinant yeast strains were inoculated into YPD medium and cultured at 30°C and 250 r / min for 24 h. The YPD culture was transferred to BMGY liquid medium and cultured at 30°C and 250 r / min. After centrifugation, the cell pellet was resuspended in BMMY medium and induced at 30°C and 250 r / min. 100% methanol was added every 24 h and cultured for 48-120 h. Take 1 mL of the induced supernatant, add trichloroacetic acid, and mix by inverting. Centrifuge at 12000 r / min for 10 min, discard the supernatant, and carefully remove the residual waste liquid. Add acetone, wash thoroughly, centrifuge at 12000 r / min for 10 min, discard the supernatant, and repeat this step twice. After the treated sample is mixed with 5× SDS loading buffer, boil for 5 min, and immediately place on ice to cool to room temperature.

[0092] SDS-PAGE was used to detect the expression level of chicken recombinant dual cytokines. After preparing the polyacrylamide gel, 10 μL of the treated sample was added to the gel wells. First, the sample was concentrated in the stacking gel by 80V constant voltage electrophoresis. After the sample entered the lower gel, the sample was dispersed by 120V constant voltage electrophoresis. The sample was stopped after bromophenol blue migrated out of the gel block. The polyacrylamide gel containing the sample was taken out and placed in a container containing 50 mL of distilled water. Wash 3-5 times to remove impurities such as SDS in the gel. Add 10 mL of Coomassie Brilliant Blue rapid staining solution and stain on a shaker at room temperature for 60 minutes. Then add 50 mL of deionized water and decolorize on a shaker. Observe the results after decolorization (such as Figure 4 The results showed that the recombinant yeast No. 2 could express a large amount of chicken recombinant dual cytokines, and its expression was 3-5 times that of other groups. Therefore, the recombinant yeast No. 2 was used as an engineered bacteria for producing ChIL2-IFNα fusion protein.

[0093] 2. Optimization of recombinant yeast induced expression conditions

[0094] In order to make the recombinant yeast express more efficiently, the formaldehyde concentration, induction time, temperature and other conditions for yeast strain induction expression were optimized. 0.5%, 1%, 2%, 3%, 4% and 5% methanol were set for induction experiments, and the supernatant was collected for SDS-PAGE analysis. The results showed (such as Figure 5 The protein expression level was the highest when the methanol concentration was 0.5%-2%. The target protein concentration in the supernatant was evaluated at 24h, 36h, 48h, 72h, 96h, 120h, and 144h induction time, and SDS-PAGE analysis was performed. The results showed (as shown in Figure 6 The target protein content in the supernatant of the yeast strain was the highest when the yeast strain was induced to express for 48-120 hours. The target protein was expressed by the yeast strain at 26℃, 28℃, 30℃ and 32℃ respectively and analyzed by SDS-PAGE. The results showed that ( Figure 7 ), the target protein content in the supernatant was the highest at 28-30℃. Three parallel experiments were performed for each group.

[0095] 3. Purification of Chicken Recombinant Dual Cytokines

[0096] According to the above optimized expression conditions, the recombinant yeast was induced to express the chicken recombinant double cytokine. 50 ml of supernatant was added to an ultrafiltration tube with a molecular weight cutoff of 10 kDa, and pre-cooled 1XPBS buffer was added. After centrifugation at 4 ° C 5000 rpm for 10 min, it was taken out and continued to add pre-cooled 1XPBS buffer and centrifuged for 10 min. After repeating this step 5-6 times, the protein was concentrated to about 10 ml and then stopped. Then the concentrated sample was added to the ultrafiltration tube of 50 kDa. The operation steps were the same as above. After repeating this step 5-6 times, the protein was concentrated to about 2 ml and then stopped. At the same time, 50 ml of supernatant was taken and the sample was purified according to the instructions using a Ni column (item number: SA004005, Tiandi Renhe Biotechnology Co., Ltd.) to obtain 2 ml of the target protein. The ultrafiltration concentrated sample and the Ni column treated sample (item number: SA004005) were taken for Western blot analysis. The results showed that ( Figure 8 First, both ultrafiltration and Ni column treatment removed contaminants from the sample, resulting in the purified target protein accounting for over 90% of the total protein. Second, the ultrafiltration-treated sample retained a higher concentration of the target protein, with a BCA assay yielding 2.1 mg / ml, compared to 0.8 mg / ml in the Ni column-treated sample. Therefore, ultrafiltration is a simpler and more cost-effective method for purifying the target protein, with minimal protein loss.

[0097] Example 4 Determination of Antiviral Activity of Chicken Recombinant Dual Cytokines

[0098] The cytopathic inhibition assay was used to determine the activity of chicken recombinant double cytokine in inhibiting VSV proliferation in chicken embryo fibroblasts (CEF). After CEF cells were cultured to a monolayer on a cell culture plate, chicken recombinant double cytokine was added at 4 -1 to 4 -11 After the dilution, add the cells to the cell culture plate, incubate at 37℃ for 24h, discard the supernatant, add 100TCID 50 When the positive control wells showed more than 75% lesions, the fluorescence results were observed and recorded ( Figure 9 ), the anti-VSV activity of chicken recombinant double cytokine was calculated by Reed-Muench method, and the activity was 1.08×10 7 IU / mL.

[0099] Example 5 Antiviral Experiment of Chicken Recombinant Dual Cytokine

[0100] 48 8-day-old SPF chicken embryos were divided into four groups, named 1, 2, 3, and 4. Groups 1 and 3 were inoculated with 0.1 ml of the active ingredient, 10 4 IU / mL chicken recombinant double cytokine, 24 hours after inoculation, groups 1 and 2 were inoculated with 10 1 EID 50 / ml of H9 subtype influenza virus, groups 3 and 4 were inoculated with 10 1 EID 50 / ml Newcastle disease virus. The hemagglutination titer of chicken embryos was measured at 12, 24, 48 and 72 hours. Figure 10 As shown, chicken recombinant double cytokine inhibited viral replication in chicken embryos.

[0101] Example 6 Effect of Chicken Recombinant Double Cytokine as Vaccine Adjuvant

[0102] Nine 21-day-old SPF chickens were divided into three groups, with three chickens in each group. Group 1 was immunized with the new avian bivalent vaccine + chicken recombinant double cytokine, group 2 was immunized with the new avian bivalent vaccine, and group 3 was the negative control group. Group 1 SPF chickens were injected with 0.5mL of the bivalent vaccine in the left leg muscle and 0.5mL of the chicken recombinant double cytokine in the right leg muscle; Group 2 SPF chickens were injected with 0.5mL of the bivalent vaccine in the left leg muscle; and the control group was injected with 1mL of PBS in the leg muscle. Blood was collected from the subwing vein on the 0th, 7th, 14th, and 21st day after immunization, and the serum was separated. The hemagglutination inhibition test (HI) was used to detect the average titer of NDV and H9 serum antibodies in each group of chickens. The results are shown in the figure. Figure 11 As shown, chicken recombinant double cytokine as a vaccine adjuvant significantly increased the animal antibody titer.

[0103] In summary, the present invention has obtained an engineered bacterium that efficiently produces chicken recombinant dual cytokine. This was achieved through screening using MD plates, MM plates, and different concentrations of G418. The purification process for the chicken recombinant dual cytokine is simple and cost-effective. The chicken recombinant dual cytokine is purified by ultrafiltration in two steps: at 50 kD and 10 kD, to obtain the target protein with a purity exceeding 90%.

[0104] 4. The product of the present invention can be used to treat chicken viral diseases.

[0105] 5. The product of the present invention can be used as an immune adjuvant to improve the effect of vaccines.

[0106] Although the principles of the present invention have been described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Without departing from the spirit and scope of the present invention, any obvious changes such as equivalent transformations and simple substitutions based on the technical solution of the present invention fall within the scope of protection of the present invention.

Claims

1. A chicken recombinant double cytokine encoding gene, characterized in that: The chicken recombinant dual cytokine encoding gene contains complete ChIL-2 and ChIFN-α structures.

2. The chicken recombinant dual cytokine expression gene according to claim 1, characterized in that: The chicken recombinant double cytokine encoding gene comprises the nucleotide sequences in SEQ ID No. 1 and SEQ ID No. 2 or the nucleotide sequences in SEQ ID No. 3 and SEQ ID No. 4 in the sequence listing.

3. A method for preparing the chicken recombinant double cytokine fusion protein according to claim 1, characterized in that: The method comprises: Step S1, selecting a Pichia pastoris expression system; Step S2, connecting the ChIL-2 and ChIFN-α sequences to construct a recombinant chicken dual cytokine, and optimizing the sequence according to the codon preference of Pichia pastoris to obtain the gene sequence ChIL-2-IFN-α that fused and expressed ChIL-2 and ChIFN-α; Step S3, connecting the gene sequence ChIL-2-IFN-α to the target vector to construct a recombinant vector, and transferring the constructed recombinant vector into competent cells of the target strain to obtain recombinant yeast; Step S4: using the obtained recombinant yeast to inoculate and culture, and detecting the expression level of the chicken recombinant dual cytokine in the recombinant yeast, and screening strains containing the target protein based on the expression level to obtain the desired cultured bacteria with high expression level; Step S5, multiplying and culturing the obtained cultured bacteria with high expression levels; Step S6: inoculating the cultured bacteria with high expression levels into the culture medium and inducing the cultured bacteria to produce proteins containing the recombinant chicken double cytokine.

4. The method according to claim 3, characterized in that The recombinant yeast includes the recombinant yeast with the deposit number of CCTCC M20232731, the deposit unit is China Center for Type Culture Collection, the address is Wuhan University, China, the deposit date is December 29, 2023, and it is named Pichia pastoris SDLCUSJY1 Pichia pastoris SDLCUSJY1.

5. The method according to claim 3, characterized in that Primers used to amplify ChIL-2 and ChIFN-α sequences include: ChIL-2-F:5'-ATGATGTGCAAAGTACTGATCTTT-3' ChIL-2-R:5'-TTATTTTTTGCAGATATCTCACAAA-3' ChIFN-α-F:5'-ATGGCTGGCCTGCAAGCCCACAG-3' ChIFN-α-R:5'-CTAAGTGCGCGTGTGCCTTGTGAG-3', In step S3, ChIL-2 and ChIFN-α are linked using a flexible linker, and the sequence of the flexible linker is shown as SEQ ID No. 6 in the sequence listing.

6. The method according to claim 3, characterized in that When inducing expression in step S6, 0.5%-2% methanol is added, the induction temperature is 28-30° C., and the induction expression time is 48-120 hours.

7. An application of Pichia pastoris, characterized in that: The method is used for preparing chicken recombinant double cytokines. Preferably, the Pichia pastoris is a yeast with a preservation number of CCTCC M 20232731.

8. A use of the chicken recombinant double cytokine according to claim 1, characterized in that: The chicken recombinant double cytokine is used for inoculation into poultry, thereby inhibiting the replication of viruses in the poultry.

9. The use according to claim 8, characterized in that The chicken recombinant double cytokine is used for inoculation into chickens.

10. The use according to claim 8, characterized in that The chicken recombinant double cytokine is used for inoculation into chicken embryos.

Images