Fusion protein for improving antiviral titer of prokaryotically expressed porcine interferon alpha

Abstract

The invention relates to a fusion protein for improving the antiviral titer of prokaryotically expressed porcine interferon-alpha, and belongs to the technical field of animal medicine. The fusion protein is an sIL-2-Thy alpha 1 fusion protein, and is a high-purity sIL-2-Thy alpha 1 protein obtained by connection and recombinant expression of an sIL-2 gene and a Thy alpha 1 gene, chromatographic purification by using Ni < 2 + > affinity resin, removal of a label by enzyme digestion and removal of endotoxin. The sIL-2-Thy alpha 1 fusion protein and the sIFN-alpha protein are combined into an antiviral composition, so that the inhibition effect of the sIFN-alpha on the swine vesicular stomatitis virus (VSV) can be remarkably enhanced, and a remarkable synergistic effect is achieved. The composition has unique advantages in the aspect of preventing and treating VSV infection. In addition, through innovative fusion protein design, the antiviral efficacy of interferon is improved, and the interferon has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of animal medicine, and in particular relates to a fusion protein for improving the antiviral potency of prokaryotically expressed porcine interferon alpha. Background Art

[0002] Interferon (IFN) is a class of small molecule glycoproteins produced by host cells after being infected by pathogens such as viruses, bacteria, parasites or stimulated by other inducers. It has a wide range of immunomodulatory and antiviral activities. In animal treatment, especially for viral diseases of economic animals such as pigs, interferon, as a natural immunomodulator, has been highly anticipated. However, with the deepening of research, the limitations of interferon's antiviral effect alone in animal treatment have gradually emerged, and its antiviral effect varies significantly for different types of viruses. Therefore, the application of interferon in large-scale breeding industry has been challenged in many aspects.

[0003] Vesicular stomatitis virus (VSV) is a virus that can infect a variety of livestock and belongs to the Rhabdoviridae family. VSV is a negative-strand RNA virus with a wide host range. Infection can cause symptoms similar to foot-and-mouth disease in a variety of livestock (including pigs, cattle, horses, etc.), and therefore has an important economic impact in the breeding industry. Studies have shown that VSV has a certain resistance to the inhibitory effects of interferon. For example, the matrix protein (M protein) of VSV is considered to be a key factor in the virus's inhibition of the interferon response in host cells. Therefore, how to effectively improve the therapeutic effect of interferon on VSV is a problem that needs to be solved at present. Summary of the invention

[0004] The present invention provides a fusion protein for improving the antiviral potency of prokaryotically expressed swine interferon (sIFN)-α, thereby effectively improving the effect of sIFN in inhibiting VSV.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a composition for inhibiting VSV, wherein the composition is composed of sIL-2-Thyα1 fusion protein and sIFN-α protein.

[0007] Preferably, in the composition, the concentration of the sIL-2-Thyα1 fusion protein is 0.05 mg / mL, and the concentration of the sIFN-α protein is 0.1 mg / mL, calculated by mass ratio.

[0008] Preferably, the method for preparing the sIL-2-Thyα1 fusion protein comprises the following steps:

[0009] (1) amplifying the sequence-optimized sIL-2-Thyα1 gene using the primer sequence of sIL-2-Thyα1 and inserting it into a prokaryotic expression vector to obtain a sIL-2-Thyα1 recombinant plasmid;

[0010] (2) transforming the sIL-2-Thyα1 recombinant plasmid into Escherichia coli, inducing the Escherichia coli to express sIL-2-Thyα1 using IPTG to obtain a bacterial solution;

[0011] (3) After centrifuging the bacterial solution and removing the supernatant, PBS was added to resuspend the precipitate, and after ultrasonic disruption, the supernatant was collected to obtain a sIL-2-Thyα1 mixed solution;

[0012] (4) The sIL-2-Thyα1 mixed solution was treated with Ni 2+ Purification by affinity resin chromatography column gave sIL-2-Thyα1 solution A;

[0013] (5) after removing endotoxin and concentrating sIL-2-Thyα1 solution A, enterokinase was added for enzymatic digestion to obtain sIL-2-Thyα1 solution B;

[0014] (6) sIL-2-Thyα1 solution B was treated with Ni 2+ The protein remaining on the column was purified by affinity resin chromatography and eluted with Tris-HCl buffer, and the flow-through and washing liquid were combined to obtain high-purity sIL-2-Thyα1.

[0015] Preferably, the method for preparing the sIFN-α protein comprises the following steps:

[0016] (1) amplifying the sequence-optimized sIFN-α gene using PCR primers of the sIFN-α gene, inserting the sIFN-α gene into a prokaryotic expression vector, and obtaining a sIFN-α recombinant plasmid;

[0017] (2) transforming the sIFN-α recombinant plasmid into Escherichia coli, inducing the Escherichia coli to express sIFN-α using IPTG to obtain a bacterial solution;

[0018] (3) After the bacterial solution was centrifuged to remove the supernatant, PBS was added to resuspend the precipitate, and after ultrasonic disruption, the supernatant was removed by centrifugation, and the precipitate was resuspended by inclusion body equilibration buffer, incubated at 4°C overnight, and the supernatant was collected by centrifugation to obtain a sIFN-α mixed solution;

[0019] (4) The sIFN-α mixed solution was treated with Ni 2+ After purification by affinity resin chromatography, protein renaturation and desalting were performed to obtain sIFN-α solution A;

[0020] (5) after removing endotoxin and concentrating sIFN-α solution A, thrombin is added for enzymatic cleavage to obtain sIFN-α solution B;

[0021] (6) Purify sIFN-α solution B using a cobalt affinity resin chromatography column and elute the protein remaining on the chromatography column using a Tris-HCl buffer, combine the flow-through and the rinse to obtain a high-purity sIFN-α protein.

[0022] Preferably, the sequences of the PCR primers of the sIL-2-Thyα1 gene are shown in SEQ ID NO.4 and SEQ ID NO.5;

[0023] The sequences of the PCR primers of the sIFN-α gene are shown in SEQ ID NO.6 and SEQ ID NO.7.

[0024] In a second aspect, the present invention provides an application of a composition in the preparation of a drug for inhibiting VSV, characterized in that the composition consists of a sIL-2-Thyα1 fusion protein and a sIFN-α protein;

[0025] In terms of mass ratio, in the composition, the concentration of the sIL-2-Thyα1 fusion protein is 0.05 mg / mL, the concentration of the sIFN-α protein is 0.1 mg / mL, and the mass ratio of the composition is: sIL-2-Thyα1:sIFN-α=1:2 (m:m).

[0026] Preferably, the sIL-2-Thyα1 fusion protein is prepared by the preparation method according to claim 3.

[0027] Preferably, the sIFN-α protein is prepared by the preparation method according to claim 4.

[0028] In a third aspect, the present invention improves the use of a fusion protein in the preparation of a drug for improving the anti-VSV effect of sIFN, wherein the fusion protein is a sIL-2-Thyα1 fusion protein, and the sIL-2-Thyα1 fusion protein is prepared by the preparation method described in claim 3.

[0029] The beneficial effects of the present invention are:

[0030] By using the sIL-2-Thyα1 fusion protein prepared by the present invention in combination with sIFN-α protein at a specific concentration, the present invention significantly enhances the inhibitory effect of sIFN-α on VSV, especially when the concentration of sIL-2-Thyα1 is 0.05 mg / mL and 0.1 mg / mL sIFN-α is combined, a significant synergistic inhibitory effect is produced. This provides a more effective composition for the prevention and treatment of VSV.

[0031] By fusing sIL-2 with Thyα1 and using them in combination with sIFN-α, the present invention makes innovative designs in the regulation and enhancement of protein function, so that the fusion protein has a synergistic effect in enhancing the antiviral effect of sIFN-α, solving the problem of limited antiviral efficacy of sIFN-α when used alone.

[0032] By optimizing the preparation and purification process of sIL-2-Thyα1 and sIFN-α, the present invention provides an efficient and simple preparation method, ensuring that the obtained protein has high quality, high purity, and complete functions, and is suitable for large-scale production applications.

[0033] In summary, the present invention has significant advantages and innovations in improving the therapeutic effect of anti-VSV, enhancing the function of interferon and its application in animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the PCR amplification result of 1sIL-2-Thyα1;

[0035] Figure 2 The result of double enzyme digestion of sIL-2-Thyα1;

[0036] Figure 3 The results of SDS-PAGE identification of pET 32b(+)-sIL2-Thyα1 under different IPTG induction concentrations;

[0037] Figure 4 The SDS-PAGE identification results of pET 32b(+)-sIL2-Thyα1 protein at the optimal imidazole washing and elution concentration;

[0038] Figure 5 The figure is the SDS-PAGE identification result of the eluate after sIL2-Thyα1 protease cleavage;

[0039] Figure 6 is the PCR amplification result of sIFNα;

[0040] Figure 7 This is the result of double restriction digestion of pET32b-sIFNα;

[0041] Figure 8 The SDS-PAGE identification results of pET32b-sIFNα under different IPTG induction concentrations;

[0042] Fig. 9 This is the SDS-PAGE identification result of pET32b-sIFNα protein under the optimal imidazole washing and elution concentration;

[0043] Fig.10For WB identification of sIFN-α protein;

[0044] Fig.11 is the activity test result of the control well;

[0045] Fig.12 is the activity test result of the sample well;

[0046] Fig.13 Activity test results for negative sample wells. DETAILED DESCRIPTION

[0047] Now describe in detail various exemplary embodiments of the present invention, this detailed description should not be considered as limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing a particular embodiment, and are not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the range.

[0048] Porcine thymosin α1 (sThyα1) is an acidic polypeptide composed of 28 amino acids. Since the sThyα1 gene sequence is too short, it is difficult to achieve genetic engineering recombinant expression alone, and it is generally fused with other proteins for expression. However, there are no reports on the fusion expression of porcine interleukin-2 (sIL-2) and Thyα1, and the function of the fusion expression is still unknown. Therefore, the present invention fuses porcine interleukin-2 and Thyα1 and explores its application in enhancing the antiviral effect of interferon.

[0049] Example 1

[0050] Preparation of sIL-2-Thyα1 protein

[0051] A. Construction of prokaryotic expression plasmid of sIL-2-Thyα1 gene

[0052] (1) Based on the sequence of IL2 gene SEQ ID NO.1, the sequence of thymosin α1 SEQ ID NO.2, and the sequence of connecting DNA SEQ ID NO.3, the primer sequence of sIL-2-Thy α1 was designed:

[0053] 5'-GCGGATCCCGGCTCCGACCTCTTCTTCTAC-3, SEQ ID NO.4

[0054] 5'-GCAAGCTTTCAGTTTCTCCGCTTCTTCCACC-3',SEQ ID NO.5

[0055] The sIL-2-Thyα1 gene was amplified. The PCR amplification pattern of the sIL-2-Thyα1 recombinant gene is shown in Figure 1 shown.

[0056] (2) The sIL-2-Thyα1 recombinant gene was inserted into the prokaryotic expression vector pET32b(+) to obtain the recombinant expression plasmid pET32b(+)-sIL2-Thyα1. The double restriction enzyme digestion map of sIL-2-Thyα1 is shown in the following figure: Figure 2 shown.

[0057] B. Transformation and induced expression of recombinant plasmid

[0058] (1) The constructed recombinant plasmid pET32b(+)-sIL2-Thyα1 was transformed into Rossetta TM (DE3) competent cells, and the transformed bacteria were spread on LB culture plates containing ampicillin resistance.

[0059] (2) Pick a single clone and transfer it to LB medium containing ampicillin (A+) resistance, and culture it in a shaker at 220 rpm and 37°C for 15 h.

[0060] (3) Take 4 mL of the above bacterial solution and inoculate it into 200 mL of LB medium containing A+ resistance. Cultivate it at 220 rpm and 37°C until OD600 = 0.5. Then add IPTG with a final concentration of 0.4 mM and induce it at 37°C constant temperature shaking incubator for 6 h to obtain bacterial solution.

[0061] pET 32b(+)-sIL2-Thyα1 was identified by SDS-PAGE at different IPTG induction concentrations. Figure 3 shown.

[0062] C. Soluble protein acquisition and purification

[0063] (1) Centrifuge the above bacterial solution at 6,000 rpm for 30 min at 4°C, collect the precipitate, resuspend the bacterial cells in 200 mL of 4°C pre-cooled PBS, centrifuge at 11,000 rpm for 30 min at 4°C, and discard the supernatant.

[0064] (2) Add 20 mL of pre-cooled PBS to resuspend the cells and perform ultrasonic disruption. Set the amplitude to 35%, sonicate for 3 seconds and stop for 5 seconds until the sample becomes clear and transparent. Centrifuge quickly after ultrasonication at 11,000 rpm for 30 minutes at 4°C and save the supernatant.

[0065] (3) Ni 2+The affinity resin chromatography column was first balanced with water and balance solution, the prepared protein was loaded, bound at 4°C for 12 hours, and then the column was washed with balance solution. Finally, the target protein was eluted with 50, 80, 100, 150, 200, 250, 300, 400, and 500 mmol / L imidazole eluents in sequence.

[0066] (4) The buffer solution was prepared according to the instruction manual of the nickel affinity resin, and the purified product was analyzed by SDS-PAGE.

[0067] The optimal imidazole washing and elution concentration of pET 32b(+)-sIL2-Thyα1 protein was determined by SDS-PAGE. Figure 4 shown.

[0068] D. Endotoxin Removal

[0069] (1) Add 1% Triton X-114 to the above protein solution, stir under magnetic stirring at 4°C for 60 min, and mix thoroughly; place in a 30°C water bath for 40 min, stirring occasionally.

[0070] (2) Centrifuge at 15,000 g for 15 min at 25°C and carefully remove the upper aqueous phase.

[0071] (3) Repeat steps (1) and (2) for two cycles.

[0072] E. Protein exchange, concentration and content determination

[0073] (1) Add the protein solution to the dialysis bag and clamp the dialysis clamps on both sides; place it in a beaker containing 1L of dialysis solution Tris-HCl (pH = 8.0), put it in a clean rotor, and slowly stir and dialyze at 4°C and 300 rpm. Replace the dialysis solution every 4-8 hours.

[0074] (2) Place the dialysis bag containing the protein sample in a dry container, pour PEG8000 onto the dialysis bag so that the PEG8000 completely covers the dialysis bag, place at 4°C, and concentrate to the required volume; use the BCA method to determine the protein content.

[0075] F. Protease cleavage

[0076] The required amount of enterokinase is calculated according to 500 μg / U of the protein to be digested, and the protein is digested at 25°C for 16 h. After being taken out, it is passed through a Ni2+ affinity resin chromatography column, and the flow-through is collected. It is then rinsed with 25 mM Tris-HCl, and the rinse is collected and combined with the flow-through. After concentration according to the above steps, a sIL-2-Thyα1 protein solution is obtained, and the protein concentration is determined and set aside.

[0077] The results of SDS-PAGE identification of the eluate after sIL2-Thyα1 protease cleavage are as follows Figure 5shown.

[0078] Example 2

[0079] preparation

[0080] sIFNα protein

[0081] A. Construction of prokaryotic expression plasmid of sIFN-α gene

[0082] (1) Primer sequence using sIFN-α (Genbank number: X56750.1):

[0083] F:5'-GCGGATCCGTGCGACCTGCCACAGACTC, SEQ ID NO.6;

[0084] R:5'-GCAAGCTTTCATTCTTTCTTACGCAGACG, SEQ IDNNO.7;

[0085] The sIFN-α gene was amplified, and the sIFNα PCR analysis was shown in the figure below. Figure 5 shown.

[0086] (2) The sIFN-α gene was inserted into the prokaryotic expression vector pET32b(+) to construct the recombinant expression plasmid pET32b(+)-sIFN-α. The double restriction enzyme digestion diagram of pET32b-sIFN-α is shown in the following figure. Figure 6 shown.

[0087] B. Transformation and induced expression of recombinant plasmid

[0088] (1) The constructed recombinant plasmid pET32b(+)-sIFN-α was transformed into RossettaTM (DE3) competent cells, and the transformed bacteria were spread on LB culture dishes containing A+ resistance.

[0089] (2) Pick a single clone colony and place it in LB culture medium containing A+ resistance, and culture it in a shaker at 220 rpm and 37°C for 15 h. Take 8 mL of the above bacterial solution and inoculate it in 400 mL of LB culture medium containing A+ resistance, and culture it at 220 rpm and 37°C until OD600 = 0.6, then add IPTG with a final concentration of 0.6 mM, and induce it in a constant temperature shaker at 37°C for 6 h.

[0090] The results of SDS-PAGE identification of pET32b-sIFN-α at different IPTG induction concentrations are shown in Figure 7 shown.

[0091] C. Inclusion body protein extraction and purification

[0092] (1) Centrifuge the above bacterial solution at 6,000 rpm for 30 min at 4°C and collect the precipitate.

[0093] (2) Resuspend the cells in 200 mL of 4°C precooled PBS, centrifuge at 11,000 rpm for 30 min at 4°C, and discard the supernatant.

[0094] (3) Add 20 mL of pre-cooled PBS to resuspend the cells and perform ultrasonic disruption. Set the amplitude to 35%, sonicate for 3 seconds and stop for 5 seconds.

[0095] (4) Wait until the sample becomes clear and transparent. After sonication, centrifuge quickly at 11,000 rpm for 30 min at 4°C and discard the supernatant.

[0096] (5) Resuspend the pellet in inclusion body equilibration buffer, pipette thoroughly to mix, and incubate at 4°C overnight. The next day, centrifuge at 11,000 rpm for 30 min at 4°C, collect the supernatant, and use it for purification.

[0097] Ni 2+ The affinity resin chromatography column was first balanced with water and balance solution, the prepared protein was loaded, bound at 4°C for 12 h, and then the column was washed with balance solution. Finally, the target protein was eluted with 20, 30, 40, 100, and 500 mmol / L imidazole eluents in sequence.

[0098] (6) The buffer solution was prepared according to the instruction manual of the nickel affinity resin, and the purified product was analyzed by SDS-PAGE.

[0099] The optimal imidazole washing and elution concentration of pET32b-sIFN-α protein was determined by SDS-PAGE. Figure 8 shown.

[0100] D. Inclusion body protein refolding and desalting

[0101] (1) Place 50 mL of the renaturation dilution solution in a 4°C refrigerator, slowly add 5 mL of the protein sample to be renatured, and place at 4°C for 24 h.

[0102] (2) After 24 hours, take out the prepared dialysis bag, clamp the dialysis clamp on one side, check for water leakage, add the diluted renatured protein in the previous step, clamp the dialysis clamp on the other side and place it in a beaker containing 1L of dialysis solution (pH = 8.0), put in a clean rotor, and slowly stir at 300 rpm at 4°C for dialysis. Replace the dialysis solution every 4-8 hours, and replace it 3 times in total.

[0103] E. Endotoxin Removal

[0104] (1) Add 1% Triton X-114 to the above protein solution, stir under magnetic stirring at 4°C for 60 min, and mix thoroughly; place in a 30°C water bath for 40 min, stirring occasionally.

[0105] (2) Centrifuge at 15,000 g for 15 min at 25°C and carefully remove the upper aqueous phase.

[0106] (3) Repeat steps (1) and (2) for two cycles.

[0107] F. Protein exchange, concentration and content determination

[0108] (1) Add the protein to the dialysis bag and clamp the dialysis clamps on both sides; place it in a beaker containing 1L of dialysis solution Tris-HCl (pH = 8.0), put it in a clean rotor, and slowly stir and dialyze at 4°C and 300 rpm. Change the dialysis solution every 4-8 hours.

[0109] (2) Place the dialysis bag containing the protein sample in a dry container, pour PEG8000 onto the dialysis bag so that the PEG8000 completely covers the dialysis bag, place at 4°C, and concentrate to the required volume; use the BCA method to determine the protein content.

[0110] G. Enzymatic cleavage of proteins

[0111] The required amount of thrombin was calculated based on 500 μg / U of the protein to be cleaved, and the cleavage was carried out at 25°C for 16 h.

[0112] After taking out, pass it through a cobalt affinity resin chromatography column, collect the flow-through, then rinse with 25mM Tris-HCl, collect the rinse and combine with the flow-through, and concentrate according to the above steps to obtain sIFN-α, and set aside after measuring the protein concentration.

[0113] The results of WB identification of sIFN-α protein are as follows Fig. 9 shown.

[0114] Example 3

[0115] The sIFN-α protein prepared by the present invention is used in combination with the sIL-2-Thyα1 fusion protein to examine whether the combination of the two can effectively enhance the antiviral effect of the sIFN-α protein.

[0116] (1) sIL-2-Thyα1 and sIFN-α were mixed at a concentration ratio of 0.1:1, 0.3:1, 0.5:1, 1:1, and 2:1 (0.1:1, i.e., 10 μL sIL-2-Thyα1 + 100 μL sIFN-α + 890 μL diluent, 100 μL was taken for testing; 0.3:1, i.e., 30 μL sIL-2-Thyα1 + 100 μL sIFN-α + 870 μL diluent, 100 μL was taken for testing; 0.5:1, i.e., 50 μL sIL-2-Thyα1 + 100 μL sIFN-α + 850 μL diluent, 100 μL was taken for testing; 1:1, i.e., 100 μL sIL-2-Thyα1 + 100 μL sIFN-α+800μL diluent, mix well and take 100μL for test; 2:1, 200μL sIL-2-Thyα1+100μL sIFN-α+700μL diluent, mix well and take 100μL for test. ) The concentrations of sIL-2-Thyα1 and sIFN-α stock solutions were both 1.0mg / mL, and 0.1mg / mL sIFN-α and 0.1mg / mL sIL-2-Thyα1 were used as controls;

[0117] (2) PK-15 cells and Vero cells were seeded into 96-well plates and grown to 80-90% confluence for later use;

[0118] (3) Dilute the mixed solution, sIFN-α solution and sIL-2-Thyα1 solution in step 1 by 10 times with cell maintenance medium. -1 ~10 -9 Second-rate;

[0119] (4) After washing the cells once with PBS buffer, add the protein sample diluted in step (3) to a 96-well cell culture plate, add 100 μL to each well, and inoculate 8 wells for each dilution;

[0120] (5) 24 hours later, inoculate 100 TCID 50 VSV, PRV and PEDV, negative control (the last column without interferon, only 100 TCID 50 Virus); observe for 2 days and record the results, which are calculated according to the Reed-Muench method. The results are shown in Table 1.

[0121] At the same time, the antiviral activity of sIFN-α control and sIL-2-Thyα1 control was determined.

[0122] Table 1 Test results of the effects of mixing sIL-2-Thyα1 and sIFN-α proteins at different mass ratios on the activity of various viruses

[0123]

[0124] As can be seen from Table 1, when used alone, sIFN-α has a certain inhibitory effect on the three viruses, while sIL-2-Thyα1 has no inhibitory effect.

[0125] For PRV and PEDV, mixing sIFN-α and sIL-2-Thyα1 at different concentrations had no significant effect on the antiviral activity, indicating that the addition of sIL-2-Thyα1 did not enhance the inhibitory effect of sIFN-α on PRV and PEDV viruses.

[0126] As for VSV, when the concentration of sIL-2-Thyα1 is low, the addition of sIL-2-Thyα1 increases the anti-VSV activity of sIFN-α. When the concentration of sIL-2-Thyα1 is 0.05 mg / mL, the anti-VSV activity of sIFN-α reaches the optimal level. After that, the concentration of sIL-2-Thyα1 does not increase the anti-VSV activity of sIFN-α, but instead produces a certain inhibitory effect. The possible reason is that when the concentration of sIL-2-Thyα1 is low, it can increase the sensitivity of cells to sIFN-α by stimulating certain signaling pathways of cells, so that it can better inhibit the proliferation of VSV. When the concentration of sIL-2-Thyα1 is too high, it may produce a certain inhibitory effect due to negative feedback and other mechanisms. Therefore, when 0.05 mg / mL of sIL-2-Thyα1 is used in combination with 0.1 mg / mL of sIFN-α, VSV proliferation can be significantly inhibited.

[0127] Example 4

[0128] (1) According to the same method as Example 1, sIL-1-Thyα1 protein and sIL-15-Thyα1 protein were prepared;

[0129] (2) Referring to the detection method of Example 3, it was detected whether sIL-1-Thyα1 protein and sIL-15-Thyα1 protein can also synergistically inhibit VSV virus when used in combination with sIFN-α at a ratio of 0.5:1. The results are shown in Table 2.

[0130] Table 2 Test results of the effects of sIL-1-Thyα1 protein, sIL-15-Thyα1 protein and sIFN-α protein on the activity of various viruses

[0131]

[0132] From the results in Table 2, it can be seen that when sIL-1-Thyα1 or sIL-15-Thyα1 is used in combination with sIFN-α, VSV proliferation cannot be significantly inhibited, indicating that when sIL-2-Thyα1 is replaced by sIL-1-Thyα1 or sIL-15-Thyα1, it cannot produce a significant synergistic effect when used in combination with sIFN-α. This may be due to the differences in cell surface receptors or cell signaling pathways stimulated by sIL-1-Thyα1 or sIL-15-Thyα1 and sIL-2-Thyα1, resulting in differences in synergistic effects.

Claims

1. A composition for inhibiting swine vesicular stomatitis virus, characterized in that: The composition consists of sIL-2-Thyα1 fusion protein and sIFN-α protein.

2. The composition according to claim 1, characterized in that In terms of mass ratio, in the composition, the concentration of the sIL-2-Thyα1 fusion protein is 0.05 mg / mL, the concentration of the sIFN-α protein is 0.1 mg / mL, and the mass ratio of the composition is: sIL-2-Thyα1:sIFN-α=1:2 (m:m).

3. The composition according to claim 2, characterized in that The preparation method of the sIL-2-Thyα1 fusion protein comprises the following steps: (1) amplifying the sequence-optimized sIL-2-Thyα1 gene using the primer sequence of sIL-2-Thyα1 and inserting it into a prokaryotic expression vector to obtain a sIL-2-Thyα1 recombinant plasmid; (2) transforming the sIL-2-Thyα1 recombinant plasmid into Escherichia coli, inducing the Escherichia coli to express sIL-2-Thyα1 using IPTG to obtain a bacterial solution; (3) After centrifuging the bacterial solution and removing the supernatant, PBS was added to resuspend the precipitate, and after ultrasonic disruption, the supernatant was collected to obtain a sIL-2-Thyα1 mixed solution; (4) The sIL-2-Thyα1 mixed solution was treated with Ni 2+ Purification by affinity resin chromatography column gave sIL-2-Thyα1 solution A; (5) after removing endotoxin and concentrating sIL-2-Thyα1 solution A, enterokinase was added for enzymatic digestion to obtain sIL-2-Thyα1 solution B; (6) sIL-2-Thyα1 solution B was treated with Ni 2+ The protein remaining on the column was purified by affinity resin chromatography and eluted with Tris-HCl buffer, and the flow-through and washing liquid were combined to obtain high-purity sIL-2-Thyα1.

4. The composition according to claim 3, characterized in that The preparation method of the sIFN-α protein comprises the following steps: (1) amplifying the sequence-optimized sIFN-α gene using PCR primers of the sIFN-α gene, inserting the sIFN-α gene into a prokaryotic expression vector, and obtaining a sIFN-α recombinant plasmid; (2) transforming the sIFN-α recombinant plasmid into Escherichia coli, inducing the Escherichia coli to express sIFN-α using IPTG to obtain a bacterial solution; (3) After the bacterial solution was centrifuged to remove the supernatant, PBS was added to resuspend the precipitate, and after ultrasonic disruption, the supernatant was removed by centrifugation, and the precipitate was resuspended by inclusion body equilibration buffer, incubated at 4°C overnight, and the supernatant was collected by centrifugation to obtain a sIFN-α mixed solution; (4) The sIFN-α mixed solution was treated with Ni 2+ After purification by affinity resin chromatography, protein renaturation and desalting were performed to obtain sIFN-α solution A; (5) after removing endotoxin and concentrating sIFN-α solution A, thrombin is added for enzymatic cleavage to obtain sIFN-α solution B; (6) Purify sIFN-α solution B using a cobalt affinity resin chromatography column and elute the protein remaining on the chromatography column using a Tris-HCl buffer, combine the flow-through and the rinse to obtain a high-purity sIFN-α protein.

5. The composition according to claim 4, characterized in that The sequences of the PCR primers of the sIL-2-Thyα1 gene are shown in SEQ ID NO.4 and SEQ ID NO.5; The sequences of the PCR primers of the sIFN-α gene are shown in SEQ ID NO.6 and SEQ ID NO.

7.

6. Use of a composition in the preparation of a drug for inhibiting swine vesicular stomatitis virus, characterized in that: The composition consists of sIL-2-Thyα1 fusion protein and sIFN-α protein; According to the mass ratio, in the composition, the concentration of the sIL-2-Thyα1 fusion protein is 0.05 mg / mL, and the concentration of the sIFN-α protein is 0.1 mg / mL.

7. The use according to claim 6, characterized in that: The sIL-2-Thyα1 fusion protein is prepared by the preparation method described in claim 3.

8. The use according to claim 7, characterized in that: The sIFN-α protein is prepared by the preparation method described in claim 4.

9. Use of a fusion protein in the preparation of a drug for improving the anti-swine vesicular stomatitis virus effect of porcine IFN-α, characterized in that: The fusion protein is a sIL-2-Thyα1 fusion protein, and the sIL-2-Thyα1 fusion protein is prepared by the preparation method according to claim 3.

Images