Preparation method of high-activity recombinant cat and dog interferon

Abstract

The invention discloses a preparation method of high-activity recombinant interferon for cats and dogs. The preparation method comprises the following steps: optimizing an interferon gene sequence, obtaining engineering bacteria, expressing recombinant interferon, renaturating the recombinant interferon and purifying the recombinant interferon. The preparation method has the advantages of simple production process, easiness in operation, low cost and the like, and is beneficial to clinical popularization of interferon.

Description

Technical Field

[0001] The invention relates to the field of biological products, and in particular to a method for preparing highly active recombinant cat and dog interferon. Background Art

[0002] In 1957, Issacs and Lindermann cultured inactivated influenza virus with chicken embryo chorioallantoic membrane and discovered a soluble substance that could inhibit the replication of influenza virus when acting on other chicken embryo chorioallantoic membranes. Therefore, this substance was named Interferon (IFN). Its main functions include antiviral, anti-tumor and immune regulation, which are also the three most widely used properties at present.

[0003] As a broad-spectrum antiviral agent, interferon has great value in many aspects. In particular, it is of great significance in the prevention and treatment of tumors and viral diseases. Compared with the natural interferon produced by traditional induced lymphocytes, genetically engineered interferon has the advantages of highly similar biological activity, high purity, stable product quality, easy product quality control, and suitability for large-scale production. In addition, genetically engineered interferon can be produced on a large scale.

[0004] With the improvement of living standards, people pay more attention to the prevention and treatment of pet diseases, and the effective prevention and treatment of pet viral diseases has become the focus of research. With the increase in the number of pets, the problem of cat and dog diseases has gradually become a public health issue. In 1987, Himmler et al. first started the research on canine interferon α (CaINF-α), reported the CalNF-α gene sequence (GenBank M28624.1 GI:163973), and applied for relevant patents, recombining the gene into Escherichia coli to express canine interferon α; in 1992, Nakamura et al. first cloned the cat IFN-ω (Feline IFN-ω, FeIFN-ω) gene from cat LSA-1 (thymic tumor T cell line infected by feline leukemia virus), and since then, cat interferon and canine interferon have appeared in people's vision. Because of its antiviral properties, it has great potential for treating viral diseases in cats and dogs. Both natural and recombinant interferons have strong antiviral capabilities. Recombinant cat and dog interferons have been clinically used to prevent and treat viral diseases such as canine parvovirus, canine distemper, canine viral hepatitis, feline calicivirus, feline leukemia virus, and feline immunodeficiency virus.

[0005] There are many related research reports on cat and dog interferon in China. Chinese scientists also started to study canine interferon α in the 1990s and achieved a series of research results in gene recombination, cloning, and expression of canine interferon α. ​​Among them, Xia Chun et al. conducted research on cloning and sequencing of interferon α genes in Labrador and German shepherd dogs, but there were no subsequent research reports on expression and application. Wang Yan et al. conducted a relatively systematic study on the cloning, expression, and activity determination of canine interferon α gene. The extracted and purified CaIFN-α was tested for biological activity on canine kidney cells (MDCK), and the biological activity was 5.11×10 5 U / mg, but no further research was conducted on the composition and application of canine interferon. Ren Qisheng et al. described in detail a method for preparing recombinant canine interferon α, and provided 26 compositions containing the interferon α and its use in the preparation of antiviral drugs, and applied for a patent. This patent also describes a mutant of canine interferon α, whose specific activity is 0.5 to 1×10 7 U / mg, which is 10 to 20 times higher than the specific activity of recombinant canine interferon α; Chen Huangshi et al. constructed a strain that can efficiently express recombinant canine interferon α, with an expression level of more than 40% and a biological activity of 4×10 7 U / mg. Chinese scientists Liu Wenjun and others disclosed a new cat ω interferon and its encoding gene, and reported its application in the preparation of drugs against feline leukemia virus, FIV or parvovirus infection. Xu Yigang and others disclosed an interferon, its encoding gene and its application in antiviral aspects, and reported that its effective antiviral concentration was 0.7mg / ml, laying the foundation for the further development and application of cat interferon products. Liao Hong and others disclosed a production process of genetically engineered bacteria expressing cat ω interferon, and the obtained cat ω interferon had an anti-VSV specific activity of 1.42×10 7 Gao Xiaoping et al. disclosed a recombinant cell line that efficiently and stably expresses feline interferon-ω2 (FeIFN-ω2). The FeIFN-ω produced by the obtained recombinant cell line has strong antiviral biological activity, which can reach 6.3×

[0006] 10 8 U / mg, and its expression level after 50 consecutive passages was approximately 1.282 g / L.

[0007] In the existing technology, cat and dog interferons have defects such as low expression, low purity, and low activity, as well as low yield and low preparation efficiency in the existing process. Therefore, they are expensive and their application range is still limited. For cat interferon, although FeIFN-ω has been used internationally for many years, so far, there are no FeIFN-ω products with approved approval numbers in my country. As for canine interferon, the results of a market survey show that only the recombinant canine α-interferon (lyophilized type) applied by Tianjin Ruipu Biological and Beijing Zhongke Baike has been approved as a new veterinary drug by the Ministry of Agriculture and Rural Affairs in 2019. In the current foreign situation, only the French Virbac company is producing pet interferon, but the product is not on the market in China, and the market gap is huge. Summary of the invention

[0008] The purpose of the present invention is to provide a method for preparing highly active recombinant cat and dog interferons.

[0009] The innovation of the present invention lies in the advantages of simple production process, easy operation, low cost, etc., which is conducive to the clinical promotion of interferon.

[0010] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is:

[0011] A method for preparing highly active recombinant cat and dog interferon comprises the following steps:

[0012] (1) Optimizing the interferon gene sequence: taking the raw material prokaryotic expression vector pBV220, the interferon gene sequence and the host bacteria, wherein the interferon is feline interferon ω or canine interferon α, using a bioinformatics website and biosoftware to analyze the codon preference of the host bacteria, adjust the secondary structure of the interferon RNA, and optimize the interferon gene sequence to obtain an optimized interferon gene sequence;

[0013] (2) Obtaining engineered bacteria: Using the prokaryotic expression vector pBV220 as the backbone vector, inserting a T7 promoter upstream of the pBV220 multiple cloning site and a hexahistidine fusion tag coding sequence (6×His-Tag) upstream or downstream to obtain the vector pJSL711, fusing and cloning the optimized interferon gene sequence into the vector pJSL711 to obtain a recombinant plasmid, transferring the recombinant plasmid into the host bacteria, and obtaining an engineered bacteria containing the interferon gene recombinant plasmid through colony screening and gene sequencing;

[0014] (3) Expression of recombinant interferon: activating the engineered bacteria and inducing fermentation to obtain bacterial cells, breaking the bacterial cells to obtain inclusion bodies, and then dissolving the inclusion bodies with a second denaturant to obtain an inclusion body solution;

[0015] (4) Renaturation of recombinant interferon: renaturing the inclusion body solution with arginine and an oxidation-reduction agent to obtain a renatured protein solution;

[0016] (5) Purification of recombinant interferon: The refolded protein solution is purified by ion exchange chromatography to obtain the recombinant interferon stock solution.

[0017] Furthermore, the host bacteria in step (2) is Escherichia coli, Bacillus subtilis, Streptomyces or yeast.

[0018] Furthermore, the method used for insertion into the pBV220 multiple cloning site in step (2) is sticky end ligation, blunt end ligation, artificial linker method or homologous polyadenylation tail ligation method.

[0019] Furthermore, the induction method of inducing fermentation in step (3) is temperature induction or chemical induction, and the fermentation density is OD600=7-11.

[0020] Furthermore, in the step (3), the bacterial cells are broken and then centrifuged and washed to obtain inclusion bodies. The first denaturant is used for washing. The first denaturant contains urea, Tirs-Hcl, EDTA, Nacl and Trition-100. The concentration of urea is 1 to 3 mol / L, and the pH of the first denaturant is 8 to 9.5.

[0021] Furthermore, in step (3), the second denaturant comprises urea or guanidine hydrochloride, Tirs, EDTA and β-ME, the concentration of urea or guanidine hydrochloride is 7-9 mol / L, and the pH of the second denaturant is 8-9.5.

[0022] Furthermore, in step (4), the concentration of arginine is 0.2-0.6 mol / L, the oxidant in the redox agent is oxidized glutathione or oxidized cysteine; the reducing agent in the redox agent is β-mercaptoethanol (β-ME), dithiothreitol (DTT), dithioerythritol or reduced cysteine.

[0023] Furthermore, the ion exchange medium in the ion exchange chromatography in step (5) is Q Sepharose FF, Q Agarose HP or an anion exchange medium.

[0024] The beneficial effects of the present invention are:

[0025] 1. The present invention obtains a highly efficient prokaryotic expression vector that can efficiently express and quickly purify the target protein by fusing the T7 promoter and the hexahistidine fusion tag coding sequence (6×His-Tag) and cloning them into the vector pBV220.

[0026] 2. The present invention achieves efficient expression of recombinant interferon (target protein) by analyzing the codon preference of the host bacteria, adjusting the secondary structure of interferon RNA, optimizing the original codons, optimizing the 5' end start region, and recombining with the efficient prokaryotic expression vector pJSL711. After fermentation, the expression amount of the target protein accounts for about 41%-43% of the total bacterial protein.

[0027] 3. The present invention obtains high-expression, high-purity and high-activity recombinant interferon by optimizing the preparation process, such as optimizing the formula of the crushing and washing solution, optimizing the formula of the renaturation and purification solution, selecting the best ion exchange filler, etc. The purity can reach more than 95%, and the antiviral activity can reach

[0028] 3.16~4.52×10 10 IU / mg, and its activity is superior to the existing prokaryotic expressed recombinant interferon.

[0029] 4. The present invention has the advantages of simple production process, easy operation, low cost, etc., which is conducive to the clinical promotion of interferon. In particular, the anion exchange filler is used, pH adjustment is not required, protein will not precipitate to cause protein loss or even deformation, and heat source can be removed. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the optimized canine interferon α gene sequence in Example 4.

[0031] Figure 2 This is a diagram showing the electrophoresis detection results of the canine interferon α gene after PCR amplification in Example 4.

[0032] Figure 3 Schematic diagram of the structure of the recombinant plasmid pJSL711-CaIFN-α in Example 4.

[0033] Figure 4 This is the electrophoresis result of the recombinant plasmid pJSL711-CaIFN-α in Example 4 after double digestion with restriction endonucleases (BamHI and EcoRI).

[0034] Figure 5 This is a diagram showing the sequencing verification result of the recombinant plasmid pJSL711-CaIFN-α in Example 4.

[0035] Figure 6 This is a graph showing the protein expression results of recombinant canine interferon α before and after purification in Example 4.

[0036] Figure 7 This is the optimized feline interferon ω gene sequence in Example 5.

[0037] Figure 8 This is a diagram showing the electrophoresis detection results of the feline interferon ω gene after PCR amplification in Example 5.

[0038] Fig. 9 Schematic diagram of the structure of the recombinant plasmid pJSL711-FeIFN-ω in Example 5.

[0039] Fig.10 This is the electrophoresis result of the recombinant plasmid pJSL711-FeIFN-ω in Example 5 after double digestion and identification with restriction endonucleases (BamHI and BglII).

[0040] Fig.11 This is a diagram showing the sequencing verification result of the recombinant plasmid pJSL711-FeIFN-ω in Example 5.

[0041] Fig.12 This is a graph showing the protein expression results of the recombinant feline interferon ω before and after purification in Example 5. DETAILED DESCRIPTION

[0042] The technical scheme in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Embodiment 1: A method for preparing a highly active recombinant cat and dog interferon, comprising the following steps:

[0043] (1) Optimizing the interferon gene sequence: taking the raw material prokaryotic expression vector pBV220, the interferon gene sequence and the host bacteria, the interferon is feline interferon ω, using the bioinformatics website and biological software to analyze the preference of the host bacteria codons and adjust the secondary structure of the interferon RNA, optimize the interferon gene sequence to obtain the optimized interferon gene sequence; using SignalP-5.0

[0044] (http: / / www.cbs.dtu.dk / services / SignalP / ) was used to predict the interferon signal peptide. According to the codon preference of the host bacteria, the original signal peptide was removed and the gene sequence was optimized. The endonuclease sites BamHI and BglII were introduced at the 5′ and 3′ ends respectively using Snapgene2.3.2 software. The functional domain was predicted using SMART (http: / / smart.embl-heidelberg.de / ) and the RNA secondary structure was adjusted.

[0045] (2) Obtaining engineered bacteria: Take the raw material prokaryotic expression vector pBV220, interferon gene sequence, and host bacteria, the host bacteria is Escherichia coli. Use the prokaryotic expression vector pBV220 as the backbone vector, insert the T7 promoter upstream and the hexahistidine fusion tag coding sequence (6×His-Tag) upstream into the pBV220 multiple cloning site by sticky end ligation method to obtain the vector pJSL711, fuse and clone the optimized interferon gene sequence into the vector pJSL711 to obtain the recombinant plasmid, transfer the recombinant plasmid into the host bacteria, and obtain the engineered bacteria containing the optimized interferon gene recombinant plasmid through colony screening and gene sequencing;

[0046] (3) Expression of recombinant interferon: The engineered bacteria were activated and then induced to ferment to obtain bacterial cells. The induction method of induced fermentation was temperature induction, and the fermentation density was OD600 = 7. The bacterial cells were broken, and then centrifuged and washed to obtain inclusion bodies. The No. 1 denaturant was used for washing. The No. 1 denaturant contained urea, Tris-HCl, EDTA, Nacl and Trition-100. The concentration of urea was 1 mol / L, the concentration of Tris-HCl was 50 mmol / L, the concentration of EDTA was 1 mmol / L, the concentration of NaCl was 0.5 mmol / L, the concentration of TritonX-100 (volume concentration) was 0.1%, and the pH of the No. 1 denaturant was 8. The inclusion bodies are then dissolved with denaturant No. 2 to obtain an inclusion body solution; denaturant No. 2 contains urea, Tris-HCl, EDTA and β-ME, the concentration of urea is 7 mol / L, the concentration of Tris-HCl is 50 mmol / L, the concentration of EDTA is 1 mmol / L, the concentration of β-ME (volume concentration) is 1%, and the pH of denaturant No. 1 is 8.

[0047] (4) Renaturation of recombinant interferon: The inclusion body solution is renatured with arginine and an oxidation-reduction agent to obtain a renatured protein solution; the arginine concentration is 0.2 mol / L, the oxidant in the oxidation-reduction agent is oxidized glutathione; and the reducing agent in the oxidation-reduction agent is β-mercaptoethanol (β-ME).

[0048] (5) Purification of recombinant interferon: The refolded protein solution was purified by ion exchange chromatography to obtain a recombinant interferon stock solution. The ion exchange medium in the ion exchange chromatography was Q Sepharose FF.

[0049] Example 2: A method for preparing highly active recombinant feline and canine interferons, comprising the following steps:

[0050] (1) Optimizing the interferon gene sequence: taking the raw material prokaryotic expression vector pBV220, the interferon gene sequence and the host bacteria, the interferon is canine interferon α, using the bioinformatics website and biological software to analyze the preference of the host bacteria codons and adjust the secondary structure of the interferon RNA, optimize the interferon gene sequence to obtain the optimized interferon gene sequence;

[0051] (2) Obtaining engineered bacteria: Take the raw material prokaryotic expression vector pBV220, interferon gene sequence, and host bacteria, the host bacteria is Bacillus subtilis. Use the prokaryotic expression vector pBV220 as the backbone vector, insert the T7 promoter upstream and the hexahistidine fusion tag coding sequence (6×His-Tag) downstream of the pBV220 multiple cloning site by blunt end ligation to obtain the vector pJSL711, fuse and clone the optimized interferon gene sequence into the vector pJSL711 to obtain a recombinant plasmid, transfer the recombinant plasmid into the host bacteria, and obtain the engineered bacteria containing the interferon gene recombinant plasmid through colony screening and gene sequencing;

[0052] (3) Expression of recombinant interferon: The engineered bacteria were activated and then induced to ferment to obtain bacterial cells. The induction method of induced fermentation was chemical induction, and the fermentation density during fermentation was OD600 = 8. The bacterial cells were crushed to obtain inclusion bodies, and then the inclusion bodies were dissolved with a second denaturant to obtain an inclusion body solution; the second denaturant contained guanidine hydrochloride, Tris-HCl, EDTA and β-ME, the concentration of guanidine hydrochloride was 8 mol / L, the concentration of Tris-HCl was 50 mmol / L, the concentration of EDTA was 1 mmol / L, the concentration of β-ME (volume concentration) was 1%, and the pH of the second denaturant was 9.

[0053] (4) Renaturation of recombinant interferon: The inclusion body solution is renatured with arginine and an oxidation-reduction agent to obtain a renatured protein solution; the arginine concentration is 0.4 mol / L, the oxidant in the oxidation-reduction agent is oxidized cysteine; and the reducing agent in the oxidation-reduction agent is dithiothreitol (DTT).

[0054] (5) Purification of recombinant interferon: The refolded protein solution was purified by ion exchange chromatography to obtain a recombinant interferon stock solution. The ion exchange medium in the ion exchange chromatography was Q Agarose HP.

[0055] Example 3: A method for preparing highly active recombinant feline and canine interferons, comprising the following steps:

[0056] (1) Optimizing the interferon gene sequence: taking the raw material prokaryotic expression vector pBV220, the interferon gene sequence and the host bacteria, the interferon is feline interferon ω, using a bioinformatics website and biological software to analyze the codon preference of the host bacteria, adjust the secondary structure of the interferon RNA, and optimize the interferon gene sequence to obtain an optimized interferon gene sequence;

[0057] (2) Obtaining engineered bacteria: Take the raw material prokaryotic expression vector pBV220, interferon gene sequence, and host bacteria, the host bacteria is Streptomyces. Use the prokaryotic expression vector pBV220 as the backbone vector, insert the T7 promoter upstream and the hexahistidine fusion tag coding sequence (6×His-Tag) downstream of the pBV220 multiple cloning site using the artificial linker method to obtain the vector pJSL711, fuse the optimized interferon gene sequence and clone it into the vector pJSL711 to obtain a recombinant plasmid, transfer the recombinant plasmid into the host bacteria, and obtain the engineered bacteria containing the interferon gene recombinant plasmid through colony screening and gene sequencing;

[0058] (3) Expression of recombinant interferon: The engineered bacteria were activated and then induced to ferment to obtain bacterial cells. The induced fermentation was performed by temperature induction. The fermentation density was OD600=11. The bacterial cells are broken, and then centrifuged and washed to obtain inclusion bodies. The first denaturant is used for washing. The first denaturant contains urea, Tis-HCl, EDTA, Nacl and Trition-100. The concentration of urea is 2 mol / L, the concentration of Tris-HCl is 50 mmol / L, the concentration of EDTA is 1 mmol / L, the concentration of NaCl is 0.5 mmol / L, the concentration of TritonX-100 (volume concentration) is 0.1%, and the pH of the first denaturant is 9. The inclusion bodies are then dissolved with a second denaturant to obtain an inclusion body solution. The second denaturant is urea. The second denaturant contains urea, Tris-HCl, EDTA and β-ME. The concentration of urea is 9 mol / L, the concentration of Tris-HCl is 50 mmol / L, the concentration of EDTA is 1 mmol / L, the concentration of β-ME (volume concentration) is 1%, and the pH of the second denaturant is 9.5.

[0059] (4) Renaturation of recombinant interferon: The inclusion body solution is renatured with arginine and an oxidation-reduction agent to obtain a renatured protein solution; the arginine concentration is 0.6 mol / L, the oxidant in the oxidation-reduction agent is oxidized glutathione; and the reducing agent in the oxidation-reduction agent is dithioerythritol.

[0060] (5) Purification of recombinant interferon: The refolded protein solution is purified by ion exchange chromatography to obtain a recombinant interferon stock solution. The ion exchange medium in the ion exchange chromatography is an anion exchange medium.

[0061] Example 4: A method for preparing highly active recombinant feline and canine interferons, comprising the following steps:

[0062] (1) Optimizing the interferon gene sequence: taking the raw material prokaryotic expression vector pBV220, the interferon gene sequence and the host bacteria, the interferon is canine interferon α, using the bioinformatics website and biological software to analyze the preference of the host bacteria codons and adjust the secondary structure of the interferon RNA, optimize the interferon gene sequence to obtain the optimized interferon gene sequence;

[0063] (2) Obtaining engineered bacteria: Take the raw material prokaryotic expression vector pBV220, interferon gene sequence, and host bacteria, the host bacteria is yeast. Use the prokaryotic expression vector pBV220 as the backbone vector, insert the T7 promoter upstream and the hexahistidine fusion tag coding sequence (6×His-Tag) upstream into the pBV220 multiple cloning site by homologous polylinking method to obtain the vector pJSL711, fuse and clone the optimized interferon gene sequence into the vector pJSL711 to obtain the recombinant plasmid, transfer the recombinant plasmid into the host bacteria, and obtain the engineered bacteria containing the interferon gene recombinant plasmid through colony screening and gene sequencing;

[0064] (3) Expression of recombinant interferon: The engineered bacteria were activated and then induced to ferment to obtain bacterial cells. The induction method of induced fermentation was temperature induction or chemical induction. The fermentation density was OD600 = 9. The bacterial cells were broken, and then centrifuged and washed to obtain inclusion bodies. The No. 1 denaturant was used for washing. The No. 1 denaturant contained urea, Tris-HCl, EDTA, Nacl and Trition-100. The concentration of urea was 3 mol / L, the concentration of Tris-HCl was 50 mmol / L, the concentration of EDTA was 1 mmol / L, the concentration of NaCl was 0.5 mmol / L, the concentration of TritonX-100 (volume concentration) was 0.1%, and the pH of the No. 1 denaturant was 9.5. The inclusion bodies are then dissolved with a second denaturant to obtain an inclusion body solution; the second denaturant is urea or guanidine hydrochloride, and the second denaturant contains urea, Tris-HCl, EDTA and β-ME, the concentration of urea is 7.5 mol / L, the concentration of Tris-HCl is 50 mmol / L, the concentration of EDTA is 1 mmol / L, the concentration of β-ME (volume concentration) is 1%, and the pH of the second denaturant is 8.5.

[0065] (4) Renaturation of recombinant interferon: The inclusion body solution is renatured with arginine and an oxidation-reduction agent to obtain a renatured protein solution; the arginine concentration is 0.3 mol / L, the oxidant in the oxidation-reduction agent is oxidized cysteine; and the reducing agent in the oxidation-reduction agent is reduced cysteine.

[0066] (5) Purification of recombinant interferon: The refolded protein solution was purified by ion exchange chromatography to obtain a recombinant interferon stock solution. The ion exchange medium in the ion exchange chromatography was Q Sepharose FF.

[0067] Example 5: A method for preparing highly active recombinant feline and canine interferons, comprising the following steps:

[0068] (1) Optimizing the interferon gene sequence: taking the raw material prokaryotic expression vector pBV220, the interferon gene sequence and the host bacteria, the interferon is feline interferon ω, using a bioinformatics website and biological software to analyze the codon preference of the host bacteria, adjust the secondary structure of the interferon RNA, and optimize the interferon gene sequence to obtain an optimized interferon gene sequence;

[0069] (2) Obtaining engineered bacteria: Take the raw material prokaryotic expression vector pBV220, interferon gene sequence, and host bacteria, the host bacteria is Escherichia coli. Use the prokaryotic expression vector pBV220 as the backbone vector, insert the T7 promoter upstream and the hexahistidine fusion tag coding sequence (6×His-Tag) upstream into the pBV220 multiple cloning site by sticky end ligation method to obtain the vector pJSL711, fuse and clone the optimized interferon gene sequence into the vector pJSL711 to obtain the recombinant plasmid, transfer the recombinant plasmid into the host bacteria, and obtain the engineered bacteria containing the interferon gene recombinant plasmid through colony screening and gene sequencing;

[0070] (3) Expression of recombinant interferon: The engineered bacteria were activated and then induced to ferment to obtain bacterial cells. The induced fermentation was performed by temperature induction. The fermentation density was OD600=9. The bacterial cells are broken, and then centrifuged and washed to obtain inclusion bodies. The first denaturant is used for washing. The first denaturant contains urea, Tis-HCl, EDTA, Nacl and Trition-100. The concentration of urea is 1.5 mol / L, the concentration of Tris-HCl is 50 mmol / L, the concentration of EDTA is 1 mmol / L, the concentration of NaCl is 0.5 mmol / L, the concentration of TritonX-100 (volume concentration) is 0.1%, and the pH of the first denaturant is 8.5. The inclusion bodies are then dissolved with a second denaturant to obtain an inclusion body solution. The second denaturant is urea. The second denaturant contains urea, Tris-HCl, EDTA and β-ME. The concentration of urea is 8 mol / L, the concentration of Tris-HCl is 50 mmol / L, the concentration of EDTA is 1 mmol / L, the concentration of β-ME (volume concentration) is 1%, and the pH of the second denaturant is 9.5.

[0071] (4) Renaturation of recombinant interferon: The inclusion body solution is renatured with arginine and an oxidation-reduction agent to obtain a renatured protein solution; the arginine concentration is 0.2 mol / L, the oxidant in the oxidation-reduction agent is oxidized glutathione; and the reducing agent in the oxidation-reduction agent is β-mercaptoethanol (β-ME).

[0072] (5) Purification of recombinant interferon: The refolded protein solution was purified by ion exchange chromatography to obtain a recombinant interferon stock solution. The ion exchange medium in the ion exchange chromatography was Q Sepharose FF.

[0073] The bacterial cells in step (2) of Examples 1 to 5 were subjected to protein electrophoresis to detect the protein content of A;

[0074] In Examples 1 to 5, A is 42.6%, 42.2%, 41.7%, 41.3% and 43.1%, respectively.

[0075] The activity of the recombinant interferon stock solution in step (4) of Examples 1 to 5 was tested and the values ​​were all B;

[0076] In Examples 1 to 4, B is 3.16×10 10 IU / mg, 3.34×10 10 IU / mg, 4.29×10 10 IU / mg, 3.88×10 10 IU / mg, 4.52×10 10 IU / mg.

[0077] According to the results in the above embodiments, compared with the technology in the existing patents and the quality of interferon obtained by production, our technology can more quickly obtain interferon with higher expression and higher activity. The described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing highly active recombinant cat and dog interferon, characterized in that: The following steps are involved: (1) Optimizing the interferon gene sequence: taking the raw material prokaryotic expression vector pBV220, the interferon gene sequence and the host bacteria, wherein the interferon is feline interferon ω or canine interferon α, using a bioinformatics website and biosoftware to analyze the codon preference of the host bacteria, adjust the secondary structure of the interferon RNA, and optimize the interferon gene sequence to obtain an optimized interferon gene sequence; (2) Obtaining engineered bacteria: inserting a T7 promoter upstream of the pBV220 multiple cloning site and a hexahistidine fusion tag coding sequence (6×His-Tag) upstream or downstream to obtain the vector pJSL711, fusing and cloning the optimized interferon gene sequence into the vector pJSL711 to obtain a recombinant plasmid, transferring the recombinant plasmid into the host bacteria, and obtaining engineered bacteria containing the interferon gene recombinant plasmid through colony screening and gene sequencing; (3) Expression of recombinant interferon: activating the engineered bacteria and inducing fermentation to obtain bacterial cells, breaking the bacterial cells to obtain inclusion bodies, and then dissolving the inclusion bodies with a second denaturant to obtain an inclusion body solution; (4) Renaturation of recombinant interferon: renaturing the inclusion body solution with arginine and an oxidation-reduction agent to obtain a renatured protein solution; (5) Purification of recombinant interferon: The refolded protein solution is purified by ion exchange chromatography to obtain the recombinant interferon stock solution.

2. The method for preparing highly active recombinant cat and dog interferon according to claim 1, characterized in that: The host bacteria in step (2) is Escherichia coli, Bacillus subtilis, Streptomyces or yeast.

3. The method for preparing highly active recombinant cat and dog interferon according to claim 1, characterized in that: The method used for insertion into the pBV220 multiple cloning site in step (2) is sticky end ligation, blunt end ligation, artificial linker method or homologous polyadenylation tail ligation method.

4. The method for preparing highly active recombinant cat and dog interferon according to claim 1, characterized in that: The induction method of inducing fermentation in step (3) is temperature induction or chemical induction, and the fermentation density OD 600 =7~11.

5. The method for preparing highly active recombinant feline and canine interferon according to claim 1, characterized in that: In the step (3), the bacterial cells are broken and then centrifuged and washed to obtain inclusion bodies. The first denaturant is used for washing. The first denaturant contains urea, Tirs-Hcl, EDTA, Nacl and Trition-100. The concentration of urea is 1-3 mol / L. The pH of the first denaturant is 8-9.

5.

6. The method for preparing highly active recombinant feline and canine interferon according to claim 1, characterized in that: In step (3), the second denaturant comprises urea or guanidine hydrochloride, Tirs, EDTA and β-ME, the concentration of urea or guanidine hydrochloride is 7-9 mol / L, and the pH of the second denaturant is 8-9.

5.

7. The method for preparing highly active recombinant feline and canine interferon according to claim 1, characterized in that: In the step (4), the concentration of arginine is 0.2-0.6 mol / L, the oxidant in the oxidation-reduction agent is oxidized glutathione or oxidized cysteine; the reducing agent in the oxidation-reduction agent is β-mercaptoethanol (β-ME), dithiothreitol (DTT), dithioerythritol or reduced cysteine.

8. The method for preparing highly active recombinant feline and canine interferon according to claim 1, characterized in that: The ion exchange medium in the ion exchange chromatography in step (5) is Q Sepharose FF, Q Agarose HP or an anion exchange medium.

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