Fusion protein of sheep interferon alpha and sheep interleukin 2 and preparation method thereof

Abstract

The invention belongs to the technical field of protein engineering, and particularly relates to a sheep alpha interferon and sheep interleukin 2 fusion protein and a preparation method thereof. The preparation method comprises the following steps: connecting a ShIFNalpha-IL2 fragment to a pET32a vector to obtain a recombinant plasmid pET32a-ShIFNalpha-IL2; transforming into an E.coli BL21 competent cell, and inducing expression; and centrifuging, collecting the strain, resuspending, carrying out ultrasonic treatment, injecting inclusion body BB, completely draining, injecting the supernatant filtrate, completely draining, injecting inclusion body BB, washing, and eluting the target protein in the column by using inclusion body EB to obtain the ShIFNalpha-IL2 recombinant protein. Through SDS-PAGE electrophoresis detection, separation and purification of the fusion protein of the sheep interferon alpha and the sheep interleukin 2 are realized, and the target protein with relatively high purity is obtained. The fusion protein also has a high expression level, can effectively prolong the retention time of interferon in vivo, and improves the half-life period of interferon.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of protein engineering, and particularly relates to a sheep alpha interferon and sheep interleukin 2 fusion protein and a preparation method. BACKGROUND

[0002] China is a large sheep country (about 350 million sheep are slaughtered annually, accounting for 50% of the global slaughter volume), and the sheep industry plays an important role in agriculture and rural economy. However, due to uncertain factors such as infectious diseases, sheep production is in a non-stable development state, and it also poses a threat to food safety and public health safety. With the advent of the era of "antibiotic ban, reduction, and limitation" on the feed and breeding sides, green and efficient biological veterinary drugs will become the best choice for the prevention and control of sheep infectious diseases. Sheep composite cytokine is a recombinant fusion protein of interferon alpha (IFNα) and interleukin 2 (IL2) expressed by genetic engineering technology, which is a new type of biological veterinary drug that is green, efficient, and can replace antibiotics, and can be used for sheep antibiotic-free breeding and disease prevention and control. The protein has the functions of interferon a and interleukin 2, among which interferon a has antiviral and immune regulation functions; interleukin 2 has immune promotion function. In theory, the expression of the two in series can be used to prevent and control the currently prevalent sheep viral diseases, and can reduce production costs. Based on this, the team optimized the codons of sheep IFNα and IL2, constructed a sheep IFNα and IL2 recombinant fusion gene engineering strain, and expressed a sheep IFNα and IL2 recombinant fusion protein.

[0003] 1. Interferon has antiviral, immune regulation, and other functions.

[0004] Interferon (IFN) is a type of active factor produced by a specific cellular response after being stimulated by external factors such as viral infection. Interferon belongs to the cytokine family and is a type of highly active and multifunctional glycoprotein, which has three types: type I, type II, and type III. Type I interferon includes IFN-α, IFN-β, IFN-ε, IFN-ω, IFN-κ, IFN-δ, IFN-τ, and IFN-ζ; type II interferon only includes IFN-γ; and type III interferon includes IFN-λ, which contains four subtypes: IFN-λ1, IFN-λ2, IFN-λ3, and IFNλ4.

[0005] 1.1 Antiviral activity

[0006] Type I, II, and III interferons all have antiviral effects, but type I interferons are often more potent than type II. Following viral infection, type I interferons are the first genes induced. They not only induce the synthesis of antiviral proteins to eliminate the virus, but also act on uninfected cells to activate the expression of related genes, rapidly placing the body in an antiviral defense state and thus establishing a broad immune defense system. Interferons can activate the expression of a series of related genes that collectively mediate antiviral effects. Three major pathways of IFN-induced antiviral mechanisms have been extensively studied: the PKR pathway, the OAS pathway, and the Mx protein pathway. OAS, PKR, and Mx I are all members of the ISG family and are natural antiviral genes. The induction of IFN expression following viral infection activates their gene transcription and the synthesis of antiviral proteins, playing a crucial role in combating viral infection.

[0007] 1.2 Immunomodulatory activity

[0008] In terms of immune regulation, type II interferon plays a more prominent role. Type II interferon can enhance the expression of MHC class II molecules, increase the recognition ability of antigen presenting cells, induce cytotoxic T cells to activate specific immunity, and improve the body's immune response ability. [1] Type I interferons inhibit the expression of MHC class II molecules by cells. Type I interferons stimulate tumor cells and virus-infected cells to express MHC class I molecules, making these cells more susceptible to elimination by the immune system.

[0009] 2. Interleukin-2 has functions such as immune regulation.

[0010] IL-2 is mainly produced by CD4 + T cells produce IL-2 receptors, while CD4 + and CD8 + It is expressed by T cells and other cell populations. With the deepening of IL-2 research, it has been revealed that IL-2 plays a crucial role in the body's immune regulation. Secreted by mitogen-activated lymphocytes, it supports lymphocyte growth in culture and enhances natural killer (NK) activity in adult animals. IL-2 is species-specific. The porcine IL-2 gene is 465 bp long with a molecular weight of approximately 17.24 kJ. The porcine IL-2 gene sequence has high homology with humans, cattle, and dogs, but low homology with chicken.

[0011] IL-2 acts as an autocrine and paracrine mediator, participating in the activation of lymphocytes and natural killer cells [And the proliferation and differentiation of other cells. In addition, other various cytokines are also affected by IL-2, such as affecting the expression and release of IFNγ, TGFβ, TNFα and other immune-related factors, and it is well known that IFNγ acts as the only type II interferon and plays an immunomodulatory role; the increase of TGFβ secretion has important influence on the growth, differentiation and immune function of cells, and plays a synergistic role with IL-2. At the same time, IL-2 can improve the immune state of the body by regulating the immune response of the cells, and ensure the homeostasis of the body. SUMMARY

[0012] The present application introduces the biological functions of IFN-α and IL-2, and focuses on the application and future development direction in the sheep industry. The present application refers to the sheep alpha interferon (accession number: X59067.1) and sheep interleukin 2 (accession number: NM_001009806) gene sequences in GenBank, first, the signal peptide sequence is removed, the mature protein gene sequence is retained and codon optimization is carried out, the target gene and the recombinant plasmid are synthesized; the recombinant plasmid is transformed into BL21 competent cells and then fermented and expressed, finally the sheep alpha interferon and sheep interleukin 2 fusion protein are obtained, and the solubility of the fusion protein is identified and separated and purified, it is determined that the fusion protein is expressed in the form of inclusion body, and the molecular weight is 52.1KDa, which provides a reference scheme for the prevention and treatment of sheep infectious diseases.

[0013] The present application provides a preparation method of sheep alpha interferon and sheep interleukin 2 fusion protein, comprising the following steps:

[0014] (1) the ShIFNα-IL2 fragment with the sequence shown in SEQ ID NO. 3 is connected to the thrombin cleavage site and the Hind III enzyme cutting site of the pET32a vector, to obtain the recombinant plasmid pET32a-ShIFNα-IL2;

[0015] (2) the recombinant plasmid obtained in step (1) is transformed into E.coli BL21 competent cells, and is fermented and cultured in LB medium containing ampicillin to the logarithmic phase, and the final concentration of 0.5mmol / L IPTG is added to induce expression;

[0016] (3) centrifuging the bacteria liquid after induction expression in step (2), collecting the bacteria strains, resuspending, ultrasonic, injecting about 5 times column volume of inclusion body BB into the column after secondary centrifugation, washing the impurities not hung on the column, injecting the filtrate of the supernatant after secondary centrifugation into the column after the flow of inclusion body BB is exhausted, injecting about 5 times column volume of inclusion body BB into the column after the supernatant is exhausted, and the flow rate is the same as that of the filtrate of the supernatant after secondary centrifugation, and the impurities not hung on the column are washed; after the flow of inclusion body BB is exhausted, the target protein in the column is eluted by using inclusion body EB, and the eluate is collected in two tubes at the same flow rate, and the elution is stopped after 5 times column volume is eluted at the same flow rate, and ShIFNα-IL2 recombinant protein is obtained.

[0017] Further, in step (1), the construction of the pET32a recombinant plasmid inserts the ShIFNα-IL2 fragment into the thrombin cleavage site and the Hind III enzyme cutting site of the pET32a vector by using a seamless cloning kit.

[0018] Further, in step (3), the induction expression conditions are as follows: 2% inoculation amount is inoculated into the fermentation medium, 37℃ culture is carried out for 4h, then IPTG is added, and 30℃, 200rpm induction is carried out for 4h; wherein, the ultrasonic conditions are as follows: output power 60%, ice bath operation 20min.

[0019] Further, in step (3), the flow rate is 1-2cm / min.

[0020] The application provides a recombinant plasmid pET32a-ShIFNα-IL2.

[0021] The application provides an engineering bacteria, which is obtained by transforming the recombinant plasmid into E.coli BL21 competent cells.

[0022] The application provides a goat α-interferon and goat interleukin 2 fusion protein, which is obtained by expression and purification of the engineering bacteria through step (3).

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The application aims to connect and recombine the goat α-interferon and goat interleukin 2 genes by genetic engineering recombination technology, construct a recombinant E.coli engineering bacteria capable of simultaneously expressing the two polypeptides, the expression product of the engineering bacteria has the double immunoregulatory functions of goat α-interferon and goat interleukin 2, and is expected to more effectively enhance the immune function of animals, resist the invasion of pathogenic microorganisms, prevent the occurrence of livestock and poultry diseases, and improve the breeding benefit; in addition, the preparation of the recombinant protein can also provide a new candidate drug for the diagnosis and treatment of livestock and poultry diseases, and has important scientific significance and application value. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1Figure 1 is a prediction model of the goat IFNα-IL2 fusion protein in Example 1;

[0026] Figure 2 Figure 2 is a cloning map of the goat IFNα-IL2 fusion gene in Example 1;

[0027] Figure 3 Figure 3 is a map of pET32a-ShIFNα-EAAK-IL2 in Example 1;

[0028] Figure 4 Figure 4 is a positive strain identification map of the goat IFNα-IL2 in Example 1;

[0029] Figure 5 Figure 5 is a sequencing map of the upstream and downstream connection sites of pET32a-ShIFNα-IL2 in Example 1;

[0030] Figure 6 Figure 6 is a protein expression identification of the goat IFNα-IL2 positive strain in Example 1; wherein, M: protein Marker; 1-3: goat IFNα-IL2 fusion protein;

[0031] Figure 7 Figure 7 is a solubility identification map of the goat IFNα-IL2 fusion protein in Example 1; wherein, M: protein Marker; 1: whole bacteria; 2: precipitation; 3: supernatant. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0033] 1、The instruments, reagents, solutions and culture media used in the following examples and experimental examples of the present patent are shown in Table 1, Table 2 and Table 3:

[0034] Table 1 Instruments

[0035]

[0036] Table 2 Reagents

[0037]

[0038]

[0039] Table 3 Solutions and Culture Media

[0040]

[0041]

[0042] 2、Plasmids and Strains

[0043] The prokaryotic expression vector pET-32a(+) was preserved by the Veterinary Biological Products Laboratory of Hebei Agricultural University; BL21 competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0044] Example 1

[0045] 1. Plasmid vector design and primer synthesis

[0046] With reference to the OviIFN-α sequence (accession number: X59067.1) and the OviIL-2 gene sequence (accession number: NM_001009806) in GenBank, the signal peptide was removed and codons were optimized. The OviIFN-α and OviIL-2 gene sequences were connected using a linker to obtain the ShIFNα-IL2 fusion gene. Primers were designed for amplifying the ShIFNα-IL2 fusion gene. The resulting primer sequences are shown in Table 4, where primers ShIFNα-F and ShIL2-R were used to amplify the ShIFNα-IL2 fusion gene; primers 32-F / R were used to prepare the linearized pET32a empty plasmid vector.

[0047] OviIFN-αSEQ ID NO.1:

[0048]

[0049] OviIL-2 SEQ ID NO.2:

[0050] ;

[0051] ShIFNα-EAAK-IL2 SEQ ID NO.3:

[0052]

[0053] IFN-α: 5605-6120 = 516 bases;

[0054] IL2: 6217-6621 = 405 bases;

[0055] Linker: 6121-6216 = 96 bases.

[0056] The OviIFN-α and OivIL-2 genes are connected by the linker sequence described above, which can ensure that the OviIFN-γ and OivIL-2 do not affect each other's tertiary structure, thereby ensuring that the biological activity is not affected.

[0057] Table 4 Primer sequences

[0058]

[0059] 2. Cloning of the fusion gene and construction of the recombinant plasmid

[0060] The ShIFNα-IL2 fusion gene (1017 bp) was amplified by conventional PCR using the primers ShIFNα-F and ShIL2-R in Table 4, and the results of agarose gel electrophoresis detection showed that the size of the amplified band was consistent with the expected, indicating that the ShIFNα-IL2 fusion gene was successfully amplified. Among them, the results of agarose gel electrophoresis detection are shown in Figure 2 .

[0061] The PCR amplification system (50 μL) is shown in Table 5.

[0062] Table 5 PCR amplification system

[0063]

[0064] PCR reaction program: 98°C pre-denaturation for 3 min; 98°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 10 min.

[0065] The upstream (5') and downstream (3') of the ShIFNα-IL2 fusion gene in step 2 were connected to the Thrombin Site and Hind III of the linearized pET32a empty plasmid using the Seamless Cloning Kit (Minerva Super Fusion Cloning Kit), and the recombinant sequence is shown in Figure 3 . The specific method is as follows:

[0066] (1) The pET32a empty vector was subjected to reverse PCR using the 32F and 32R primers in Table 4 to prepare the linearized pET32a empty plasmid.

[0067] (2) The ShIFNα-IL2 fusion gene fragment amplified using the ShIFN-α-F and ShIL2-R primers in Table 4 has homologous sequences at the upstream and downstream ends of the pET32a linearized vector.

[0068] (3) A seamless cloning reaction was performed using a seamless cloning kit to connect the pET32a linearized vector and the ShIFNα-IL2 recombinant fragment to obtain the pET32a-ShIFNα-IL2 recombinant plasmid.

[0069] 3. Transformation of pET32a-ShIFNα-IL2 recombinant plasmid and identification of positive strains

[0070] Add 10 μL of reaction solution to 100 μL of competent cells, mix gently by pipetting, and incubate on ice for 30 minutes; heat shock at 42°C for 60 seconds, and immediately place on ice for 5 minutes; add 1.5 mL of LB medium containing ampicillin, and culture at 37°C, 200 rpm on a shaker for 60 minutes. Spread the bacterial solution evenly on a plate containing the corresponding antibiotics, and invert it in a 37°C incubator to culture overnight; randomly pick a single colony from the LB solid medium cultured in a 37°C incubator overnight, place it in a 15 mL centrifuge tube, add 5 mL of liquid LB medium containing ampicillin, and culture at 37°C for 4 hours; use the bacterial solution after culturing at 37°C for 4 hours as a template to prepare a 20 μL PCR system, and then detect it by 1% agarose gel electrophoresis. The electrophoresis results are shown in Figure 4 .

[0071] Take the positive bacterial suspension and inoculate 100 μL into 10 mL of liquid LB at 200 rpm. After culturing for 4 hours, extract the plasmid according to the instructions of the BIOMIGA plasmid miniprep kit and send the extracted plasmid to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results of the upstream and downstream junction sites of the recombinant fragment and the pET32a vector are shown in Figure 5 .

[0072] The strain with positive results was named pET32a-ShIFN-α-IL2 and stored in a -20°C refrigerator.

[0073] 4. Inducible expression of ShIFNα-IL2 fusion protein

[0074] Take 1 mL of the bacterial suspension of the positive strain identified in step 4, add LB liquid medium containing kanamycin sulfate to 15 mL, ferment for 4 hours, and then add IPTG to induce protein expression for 4 hours;

[0075] Take the expression of the bacteria liquid 30 μL, add 4x protein loading buffer 10 μL, shake and mix, then boil in 100°C water bath for 10 minutes. Finally, the above sample is identified by SDS-PAGE for protein expression. As shown in Figure 6 Figure 1, lane M is a rainbow spectrum 130 protein marker, lane 1 is ShIFNα-IL2 fusion protein, and the band size is consistent with the expected (ShIFN-α-IL2 recombinant protein size is 52.1 kD), indicating that the recombinant gene can be normally expressed in BL21 competent cells, and the protein expression amount is considerable.

[0076] 5. Solubility analysis of ShIFNα-IL2 fusion protein

[0077] Fermentation and induction expression: the activated positive strain was inoculated into 150 mL LB medium containing ampicillin at a inoculation amount of 2%, and cultured at 37°C, 200 rpm for 4 h. Then IPTG was added to 0.5 mmol / L, and the culture was induced for protein expression at 30°C, 200 rpm for 4 h. The bacteria liquid after induction expression culture was centrifuged at 12000 rpm for 10 min. The supernatant was discarded, and the bottom mud was resuspended with PBS solution. The output power of the ultrasonic cell disrupter was adjusted to 60%, and the resuspended liquid was ultrasonically disrupted for about 20 min. The whole process was carried out in ice bath, and the sample was taken as the whole bacterial protein sample after the particles (E. coli cells) in the disrupted liquid disappeared under naked eye observation. The disrupted liquid was centrifuged at 4°C, 12000 rpm for 15 min, and the supernatant and precipitate were reserved respectively. The supernatant part was sampled as the soluble expression sample. The precipitate part was resuspended in 25 mL 1xPBS and immediately sampled as the inclusion body expression sample. The solubility was identified by SDS-PAGE;

[0078] The specific steps of solubility identification are as follows:

[0079] Sample preparation: mix 30 μL sample with 10 μL 4x protein loading buffer in a 1.5 mL centrifuge tube, then boil in boiling water bath for 10 min, and centrifuge at 5000 rpm.

[0080] Gel preparation: use PAGE gel rapid preparation kit to prepare 1 mm thick 10-hole or 15-hole protein electrophoresis gel on the gel preparation plate, with 4.5% concentrated gel, 10% or 12.5% separation gel. The gel preparation formula is shown in Table 6:

[0081] Table 6 SDS-PAGE gel configuration table

[0082]

[0083] Electrophoresis: Place the prepared gel in an electrophoresis tank and pour 1× SDS electrophoresis buffer until the liquid level in both the inner and outer tanks is higher than the glass plate. Spot the prepared sample at 10 μL / well and spot 5 μL of rainbow protein marker as a molecular weight standard. After turning on the power, adjust the voltage to 100 V and the current to 200 mA. Run the electrophoresis for about 1.5 hours until the bromophenol blue dye reaches the bottom of the gel.

[0084] Staining: After electrophoresis is completed, remove the electrophoresis gel and place it in Coomassie Brilliant Blue R-250 staining solution and slowly shake for 4 hours until the gel color is uniform with the staining solution color;

[0085] Destaining: After carefully rinsing the stained gel with water, transfer it to boiling water and simmer for 20 hours, changing the water every 10 minutes until destaining is complete. Finally, photograph the gel for analysis.

[0086] The protein expression of the engineered bacteria was analyzed according to the SDS-PAGE results. The solubility identification results are as follows Figure 7 As shown, lane 1 is the whole bacterial cell suspension after ultrasonic disruption; lane 2 is the bacterial pellet after centrifugation; and lane 3 is the supernatant after centrifugation. Lane 2 (bacterial pellet sample) shows the same protein bands as lane 1 (whole bacterial sample); while lane 3 (supernatant sample) lacks a band, indicating that the ShIFN-α-IL2 fusion protein is an inclusion body protein.

[0087] 6. Isolation and purification of fusion protein

[0088] Take 150 mL of the bacterial suspension after fermentation induction expression in step 6, centrifuge at 10,000 rpm at 4°C for 5 min, collect the bacteria, re-select to 25-30 mL with 25 mL of 1× PBS, adjust the output power of the ultrasonic cell disruptor to 60% and ultrasonically disrupt the bacterial slurry resuspension for about 20 min. The entire disruption process is carried out in an ice bath until the granularity (E. coli cells) of the disrupted liquid disappears when observed by naked eye under light. Then, centrifuge the disrupted liquid at 12,000 rpm at 4°C for 15 min;

[0089] For proteins expressed in inclusion bodies by fermentation of engineered bacteria, the supernatant after centrifugation was collected, filtered through a 0.45 μm needle filter, and then purified by nickel affinity chromatography (gravity column).

[0090] Among them, the nickel column purification method is as follows:

[0091] Inject 5 column volumes of the inclusion body BB solution into a gravity column loaded with an appropriate amount of nickel column packing to balance the nickel column. After the inclusion body BB has flowed away, inject the centrifugal supernatant filtrate. Control the linear flow rate of the solution in the packing to approximately 1-2 cm / min by adjusting the liquid replenishment rate until the filtrate has completely passed through the column.

[0092] After the supernatant is completely drained, about 5 times the column volume of inclusion body BB is injected into the column to wash away the impurities not bound to the column, at the same flow rate; after the inclusion body BB is completely drained, the target protein in the column is eluted using inclusion body EB, and the eluate is collected in two separate tubes; after 5 times the column volume is eluted at the same flow rate, the collection is stopped, and the elution is completed; after the collection is stopped, 5 times the column volume of soluble EB is used for continuous flushing, and finally, the column is flushed with 20% ethanol solution and sealed for storage;

[0093] The collected eluate is detected by SDS-PAGE electrophoresis, in which M is a rainbow broad-spectrum 130 protein marker, lane 1 is the flow-through sample collected after the supernatant is added, lane 2 is the impurity protein sample collected by elution with inclusion body BB, and lanes 3-8 are the target protein samples collected by elution with inclusion body EB. In lanes 3-8, the target protein bands are consistent with the expected (the size of the ShIFN-alpha-IL2 fusion protein is 52.1 kD), indicating that the protein obtained by elution with inclusion body EB is the ShIFN-alpha-IL2 fusion protein.

[0094] The present application uses a rigid linker to recombinantly construct a ShIFN-alpha-IL2 fusion protein by optimizing the connection peptide of sheep alpha interferon and sheep interleukin 2, as shown in Figure 1 The molecular weight of the ShIFN-alpha-IL2 fusion protein is 52.1 KDa, which is about 2.66 times higher than that of sheep alpha interferon, reducing kidney filtration to some extent and prolonging the half-life of interferon in the body; at the same time, the multi-target effect reduces the degradation caused by off-target effects, improving the stability of interferon in the body. The ShIFN-alpha-IL2 fusion protein can also activate multiple immune pathways simultaneously, achieving synergistic effect of the two, and IL-2 can enhance the killing function of T cells, and IFN-alpha can inhibit virus replication, reducing the dosage of interleukin 2 while achieving the expected therapeutic effect.

[0095] In addition, the present application successfully realizes the separation and purification of the sheep alpha interferon and sheep interleukin 2 fusion protein through SDS-PAGE electrophoresis detection, obtaining a target protein with high purity. This result not only verifies the expression and solubility of the fusion protein, but also lays a solid foundation for subsequent biological function research and clinical application.

[0096] The above-described embodiments are only descriptions of preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for preparing a fusion protein of sheep interferon-α and sheep interleukin-2, characterized in that: The following steps are involved: (1) The ShIFNα-IL2 fragment with the sequence shown in SEQ ID NO. 3 was ligated between the thrombin cleavage site and the HindIII restriction site of the pET32a vector to obtain the recombinant plasmid pET32a-ShIFNα-IL2; (2) The recombinant plasmid obtained in step (1) was transformed into E. coli BL21 competent cells, and fermented in LB medium containing ampicillin to the logarithmic phase, and IPTG was added to a final concentration of 0.5 mmol / L to induce expression; (3) The bacterial solution after induction expression in step (2) is centrifuged, the strain is collected, resuspended and sonicated, and after a second centrifugation, about 5 times the column volume of inclusion bodies BB are injected into the column to wash away impurities not hanging on the column. After the inclusion bodies BB have flowed out, the supernatant filtrate after the second centrifugation is injected. After the supernatant has flowed out, about 5 times the column volume of inclusion bodies BB are injected into the column to wash away impurities not hanging on the column. The flow rate is the same as that of the supernatant filtrate after the second centrifugation. After the inclusion bodies BB have flowed out, the target protein in the column is eluted with inclusion body EB, and the eluate is collected in a separate tube at the same time. After eluting 5 times the column volume at the same flow rate, the collection is stopped to complete the elution and obtain the ShIFNα-IL2 recombinant protein.

2. The preparation method according to claim 1, characterized in that In step (1), the pET32a recombinant plasmid was constructed by inserting the ShIFNα-IL2 fragment between the thrombin cleavage site and the HindⅢ restriction site of the pET32a vector using a seamless cloning kit.

3. The preparation method according to claim 1, characterized in that The conditions for inducing expression in step (3) are: inoculating the fermentation medium with a 2% inoculum, culturing at 37°C for 4 hours, adding IPTG, and inducing at 30°C and 200 rpm for 4 hours; wherein, the conditions for ultrasound are: output power 60%, ice bath operation for 20 minutes.

4. The preparation method according to claim 1, characterized in that The flow rate in step (3) is: 1-2 cm / min. The recombinant plasmid pET32a-ShIFNα-IL2 according to claim 1 .

6. An engineered bacterium, characterized in that: The recombinant plasmid according to claim 5 is transformed into E. coli BL21 competent cells.

7. A fusion protein of ovine interferon-α and ovine interleukin-2, characterized in that: The engineered bacteria according to claim 6 are expressed and purified in step (3) of claim 1.

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