Expression and antiviral application of bovine interferon

Abstract

The invention provides a bovine interferon alpha recombinant protein with a long half-life period, a variant protein and an application of the bovine interferon alpha recombinant protein and the variant protein in virus resistance, a key part with an antiviral effect of BoIFN alpha and a key part with a low toxicity effect of BoIFN tau are connected by using a flexible linker to construct a low-toxicity long-acting interferon, and specific amino acid of the low-toxicity long-acting interferon is subjected to site-specific mutagenesis on the basis of the low-toxicity long-acting interferon; the toxicity and the half-life period are further improved. Tests prove that the long-acting recombinant bovine interferon product prepared by the invention is an efficient, long-acting and broad-spectrum antiviral drug product, has a longer half-life period and higher biological activity than common natural bovine interferon or hybrid interferon, prolongs the half-life period, improves the biological activity, reduces the preparation cost of an interferon preparation, and is suitable for industrial production. The compound has huge potential and value of being developed into broad-spectrum antiviral drugs and veterinary clinical application.

Description

Technical Field

[0001] The invention belongs to the technical field of biological gene engineering, and specifically relates to a bovine interferon recombinant protein with a long half-life, a variant protein and the application thereof in anti-virus. Background Art

[0002] Interferon (IFN) is a class of proteins that regulate the body's immune function, have antiviral and anti-tumor activities, and are an important part of the body's defense system. According to the differences in interferon sequence, chromosomal location, acid stability and cell surface receptors, interferons are divided into type I, type II and type III. The mammalian type I interferon family is mainly induced by viruses, including IFN-α, IFN-β, IFN-ω, IFN-δ, IFN-ε, IFN-ν, IFN-τ and IFN-κ, etc. They have similar biological activities and structural compositions, and act together on a complex receptor composed of two subunits for signal transduction, exerting an antiviral effect. Type I interferons are now widely used. Type II interferons have only one member, namely IFN-γ, and its biological activity is weaker than that of type I interferons. Type III interferons IFN-λ include IL-29, IL-28A and IL-28B.

[0003] Interferon, as a protein drug for clinical application, is metabolized by the body mainly through receptor-mediated clearance, serum protease degradation and glomerular filtration. Due to the small radius of the interferon molecule, it is easier to be cleared by the body, resulting in a short half-life. Repeated injections are required during treatment, and the treatment cycle is extended. Therefore, it is particularly important to develop interferon fusion proteins and improve the pharmacokinetic characteristics of interferon to extend its half-life in the body. Although there are interferons of different parental gene sequences in the prior art that are organically combined by genetic engineering technology to form hybrid interferons, it is also verified that IFN-τ and IFN-α in the type I interferon family are constructed by hybrid interferon mode, retaining the advantages of the above two interferons, and solving the enlightenment of clinical problems, but the effect of this hybrid interferon is not very different from that of a single interferon, and further research is needed on its core structure. Summary of the invention

[0004] In order to overcome the deficiencies in the prior art, the present invention provides a recombinant bovine interferon fusion protein, wherein the fusion protein includes a key part of the antiviral effect of BoIFNα and a key part of the low toxicity effect of BoIFNτ, and the two are connected by a flexible linker;

[0005] Furthermore, the key site of the antiviral effect of BoIFNα is the first 163 amino acids at the amino terminus of the BoIFNα mature peptide, and its amino acid sequence is shown in SEQ ID NO.1;

[0006] The key site for the low toxicity of BoIFNτ is the 11 amino acids after the carboxyl end of the mature peptide of BoIFNτ, and its amino acid sequence is shown in SEQ ID NO.2;

[0007] The flexible linker is GGSGGGS;

[0008] Furthermore, the amino acid sequence of the recombinant bovine interferon fusion protein is shown in SEQ ID NO.3.

[0009] Furthermore, the present invention provides a variant protein of the above-mentioned recombinant bovine interferon fusion protein, wherein the variant protein is a protein obtained by mutating several specific amino acids in the above-mentioned fusion protein into polar amino acids, and the above-mentioned mutation improves the flexibility of the protein, while improving the isoelectric point and stability of the protein, thereby better maintaining the biological function of the protein.

[0010] Preferably, the variant protein is a protein obtained by mutating the 10th, 37th, and 119th amino acid residues (A10, A37, and A119, respectively) in BoIFNα and the third and sixth amino acid residues from the bottom (M188 and D191, respectively) in BoIFNτ;

[0011] Preferably, the above amino acid residues are mutated to arginine, lysine or histidine;

[0012] Preferably, the variant protein is a protein obtained by mutating the 10th, 37th, and 119th amino acid residues in BoIFNα (A10 / R, A37 / H, and A119 / R, respectively) and the third and sixth amino acid residues from the bottom in BoIFNτ (M188 / K and D191 / K, respectively);

[0013] Furthermore, the amino acid sequence of the variant protein is shown in one of SEQ ID NO.4-10.

[0014] Furthermore, the present invention also provides the use of the above-mentioned fusion protein or variant protein in the preparation of bovine antiviral drugs.

[0015] The present invention also provides a recombinant bovine long-acting interferon, which is prepared by mixing the fusion protein or variant protein with a freeze-dried protective agent and then freeze-drying. The freeze-dried protective agent is selected from one or a mixture of any proportion of trehalose, sucrose, lactose, glucose, raffinose, dextran, mannitol, sorbitol or xylitol, and the amount of the added freeze-dried protective agent is 0.25-0.5%.

[0016] Beneficial Effects

[0017] Based on early research, the present invention connects the key antiviral site of BoIFNα and the key low toxicity site of BoIFNτ using a flexible linker to construct a low-toxic long-acting interferon, and on this basis, site-directed mutations are performed on specific amino acids to further improve its toxicity and half-life. The experiment of the present invention proves that the long-acting recombinant bovine interferon product prepared by the present invention is a highly effective, long-acting, broad-spectrum antiviral drug product, which has a longer half-life and higher biological activity than ordinary natural bovine interferon or hybrid interferon, prolongs the half-life, improves the biological activity, and reduces the preparation cost of interferon preparations, and has great potential and value for development into a broad-spectrum antiviral drug and veterinary clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the construction of BoIFNα-τ fusion protein;

[0019] Figure 2 Cytotoxicity validation test of fusion protein;

[0020] Figure 3 In vitro stability test of fusion protein in bovine plasma;

[0021] Figure 4 In vivo stability study of fusion proteins in bovine. DETAILED DESCRIPTION

[0022] The present invention is described more clearly and completely through the following examples, but the examples described are only part of the embodiments of the present invention, not all of them. The embodiments are to help understand the present invention and should not be used to limit the scope of protection of the present invention.

[0023] Unless otherwise specified, the vectors, host bacteria, culture media, test reagents, cells, experimental animals, etc. in this application are all derived from commercial products.

[0024] Example 1 Preparation of recombinant BoIFNα-τ fusion protein

[0025] Bioinformatics analysis revealed that the key sites of BoIFNα's antiviral effect (the first 163 amino acids at the amino terminus of the mature peptide, as shown in SEQ ID NO.1, CHLPHTHSLANRRVLMLLGQLRRVSPSSCLQDRNDFAFPQEALGGSQLQKAQAISVLHEVTQHTFQLFSTEGSATTWDES LLDKLRAALDQQLTDLQACLRQEEELQGAPLLKEDSSLAVRKYFHRLTLYLQEKKHSPCAWEVVRAQVMRAFSSSTNLQESFR) and the key sites of BoIFNτ's low toxicity effect (the last 11 amino acids at the carboxyl terminus of the mature peptide, as shown in SEQ ID NO.2, CHLPHTHSLANRRVLMLLGQLRRVSPSSCLQDRNDFAFPQEALGGSQLQKAQAISVLHEVTQHTFQLFSTEGSATTWDES LLDKLRAALDQQLTDLQACLRQEEELQGAPLLKEDSSLAVRKYFHRLTLYLQEKKHSPCAWEVVRAQVMRAFSSSTNLQESFR) were NO.2 shows the potential mutation sites in ERLRMMDGDQN): homology modeling of interferon and interferon receptor was performed through online public databases, and the molecular docking structure model was obtained using Z-DOCK. The above two fragments were scanned for mutations, and the changes in the binding force between interferon and receptor before and after mutation were analyzed to screen for potential mutation sites; after analysis, the 10th, 37th, and 119th amino acid residues in BoIFNα (A10, A37, and A119, respectively) and the 3rd and 6th amino acid residues from the bottom in BoIFNτ (M188 and D191, respectively) have high mutation significance, and the antiviral activity of the fusion protein can be increased by mutation while maintaining low biological toxicity. The schematic diagram of the fusion protein structure is shown in Figure 1 shown.

[0026] According to the target gene sequence reported in Genebank, synthetic primers were designed, RNA was extracted from bovine liver tissue, and the 1-163 amino acids of the mature peptide of BoIFNα and the 10 amino acids after the carboxyl end of the mature peptide of BoIFNτ were amplified after reverse transcription using the Invitrogen reverse transcription kit. After the target gene after ligation was selected and sequenced, the PCR gel recovery product and the pET-32a plasmid were double-digested and recovered using EcoRI and XhoI restriction endonucleases, and the ligation product was transformed into Escherichia coli BL21 (DE3) competent cells, and the competent cells were spread on LB medium plates containing ampicillin for overnight culture; single colonies on the LB plates were picked for target gene PCR identification, and the positive clone bacterial plasmid was identified by double digestion of EcoRI and XhoI. The positive one indicated that the engineering bacteria was successfully constructed, and the pET-32a / BoIFNα-τ engineering bacteria were obtained after sequencing.

[0027] The primers used were:

[0028] BoIFNα upstream primer: GGTGGATCTGGTGGTGGTTCT TGCGACTTGCCAC;

[0029] BoIFNα downstream primer: CC CTCGAG GTCGATTCTGCCAAAGC

[0030] BoIFNτ upstream primer: CG GGATCC AAAGTACATAATCCTGCT

[0031] BoIFNτ downstream primers: AGAACCACCACCAGATCCACC TAGTGAATGGTAGCTTAAA.

[0032] BGI (background) was commissioned to perform site-directed mutagenesis on the above-verified fusion protein to construct a mutant protein, and the above-mentioned fusion protein was purified for standby use. The specific fusion protein name and mutation site are shown in Table 1 below:

[0033] Table 1 Nomenclature of mutant plasmids and their mutation points

[0034]

[0035] Example 2 Verification of the physiological activity of the recombinant BoIFNα-τ fusion protein

[0036] (1) Antiviral activity verification

[0037] When the MDBK cells were growing well, the cell culture medium was discarded, the cells were washed three times with 1×PBS, digested with 0.25% trypsin, and the cells were blown with 10% fetal bovine serum DMEM culture medium until they were uniform. 1×10 5 The density of cells was spread in a 96-well cell culture plate, 100 μL per well, and placed in a 37°C CO2 cell culture incubator for culture. When the cells grew to 60% (about 12 hours), the above recombinant protein was diluted 4 times in a 5% fetal bovine serum DMEM culture medium. Added to the above 96-well cell culture plate, and cultured in a 37°C CO2 cell culture incubator for 18 hours. Take the VSV with TCID50 determined, dilute it to the working concentration (100TCID50) with serum-free DMEM, take out the 96-well cell culture plate pretreated with fusion protein, discard the original cell culture medium in the plate, add the diluted virus solution, 100 μL per well, incubate at 37°C CO2 cell culture incubator for 2 hours, discard the serum-free DMEM containing the virus, add 2% fetal bovine serum DMEM 100 μL per well, and place in a CO2 cell culture incubator for culture until the virus control well without interferon is completely diseased. Repeat the experiment three times and calculate according to the Reed-Muench method. The amount of interferon contained in the dilution that protects against 50% cytopathic effect is 1 interferon unit, and the antiviral activity is calculated as shown in Table 2.

[0038] Table 1 Antiviral activity of modified interferon

[0039] Plasmid name Antiviral activity (IU / mL) Plasmid name Antiviral activity (IU / mL) BoIFNα-τ <![CDATA[6.89×10 6 ]]> BoIFNM4 <![CDATA[2.41×10 8 ]]> BoIFNM1 <![CDATA[4.31×1084 8 ]]> BoIFNM5 <![CDATA[3.68×10 8 ]]> BoIFNM2 <![CDATA[5.17×10 6 ]]> BoIFNM6 <![CDATA[4.39×10 8 ]]> BoIFNM3 <![CDATA[3.08×10 8 ]]> BoIFNM7 <![CDATA[4.54×10 8 ]]> BoIFNα <![CDATA[5.51×10 6 ]]> - -

[0040] The above results show that compared with the negative control, the above interferons and their fusion proteins can exert strong antiviral activity. The unmutated BoIFNα-τ and BoIFNM2 with only the mutation of BoIFNτ showed strong antiviral activity similar to that of BoIFNα interferon, and there was no significant difference in antiviral activity between them. Mutations on BoIFNα greatly improved its antiviral activity, but there seemed to be no obvious pattern between its different mutation points, and the specific reasons still need further study.

[0041] (2) Cytotoxicity Verification

[0042] The low cytotoxicity was detected by CCK-8 kit. MDBK monolayer cells were prepared in 96-well culture plates. When the cells grew to 70%, the cells were treated with different concentrations of recombinant proteins (the starting concentration of the nine recombinant proteins was 16 μg / mL, and then diluted 4 times in a row), incubated in a 37°C CO2 cell culture incubator for 72 hours, and then 10 μL CCK8 was added to each well, incubated in a 37°C CO2 cell culture incubator for 4 hours, and the absorbance (OD490nm) of the cells after treatment with different concentrations of recombinant proteins was measured by a microplate reader. Each treatment group was repeated 4 wells.

[0043] The results are as follows Figure 2 As shown, the relative proliferation rate of the untreated group, i.e., the culture medium-treated group, was set to 100%, and the anti-cell proliferation activity of interferons in each group was dose-dependent. At the same concentration, the relative proliferation rate of cells mutated to BoIFNτ was higher. At relatively high concentrations, the cells could still proliferate, and their dose dependence was relatively low. This shows that compared with BoIFNα, BoIFNα-τ, and BoIFNM1, mutation of BoIFNτ helps to reduce the toxicity of the fusion protein to cells. Moreover, the cytotoxicity of the mutant fusion protein involving only M188 / K is significantly lower than that of other mutations. It is possible that its advanced conformation affects its low toxicity.

[0044] (3) Verification of half-life at the cell level

[0045] Twenty cows of roughly the same weight (half male and half female) were taken, their anticoagulated blood was collected, and plasma was collected by centrifugation at 3000rpm for 5min. The plasma was mixed with PBS at a ratio of 1:1 to prepare 50% plasma. The above 9 purified recombinant proteins were added to make the final concentration of the recombinant protein in the plasma 10μg / mL. Negative plasma without interferon was set up as a control group, and the cells were incubated in a 37℃ water bath for 0, 0.25, 0.5, 1, 3, 6, 12, and 24h, respectively. The antiviral activity was detected on the MDBK-VSV system according to the above method. The experiment was repeated three times, and the cytopathic inhibition rate was recorded. The results are as follows Figure 3 As shown, the bovine negative plasma without interferon had no antiviral activity, and the antiviral activity of the other nine interferons gradually decreased with the extension of incubation time. The half-life of BoIFNα is relatively short, and it drops significantly at 6-12 hours, and the remaining activity is less than 50% after 24 hours. The half-life of BoIFNα-τ can be steadily extended to more than 24 hours, and the activity is maintained at about 50% after 24 hours of incubation. The half-life of the modified fusion protein is significantly increased, and a high activity can be maintained within 24 hours. Its half-life is significantly longer than 24 hours, and the remaining activity is still higher than 50%.

[0046] (4) Verification of drug half-life in cattle

[0047] Twenty cows of approximately the same weight (half male and half female) were selected and injected with 9 fusion proteins (2 mg / ml, 2 ml) respectively. The remaining two cows were injected with normal saline as negative control. Venous blood was collected at 0h, 6h, 12h, 24h, 36h, 48h, 72h, and 96h after injection. The blood was left to stand at 4°C for low-temperature coagulation and then centrifuged at 3000r / min for 5min. The plasma was collected by centrifugation. The antiviral activity of the remaining interferon in the plasma was detected on the MDBK-VSV system according to the above method, and the cytopathic inhibition rate was recorded.

[0048] The results are as follows Figure 4As shown in the figure, after the injection of interferon, compared with the negative group, the antiviral activity of the remaining interferon in the plasma of all positive groups reached a peak 6 hours after the injection of interferon, and then gradually decreased. There was no significant difference in the peak time of each group. Similar to the cell level, the antiviral activity of each group gradually decreased with the extension of incubation time. The decline rate of BoIFNα and BoIFNα-τ was faster, but BoIFNα-τ was significantly better than BoIFNα without any modification. It could still maintain a certain activity after 24 hours and lost its antiviral activity after 48 hours. The inactivation time of the remaining mutant fusion proteins was longer than 48 hours, among which the remaining interferon in the plasma of BoIFNM1,2,4,5,7 at 72 hours after injection still had a certain antiviral activity. BoIFNM3 and BoIFNM6 still retained antiviral activity in the plasma at 96 hours after injection. The above results prove that the half-life of the modified fusion proteins of BoIFNα and BoIFNτ can be significantly increased, and the optimization of the specific mutation points and their specific combinations still requires further in-depth research.

[0049] In order to facilitate its use, it is prepared into a recombinant bovine long-acting interferon using common operating methods in the art, and the above fusion protein is mixed with a lyophilization protective agent and then freeze-dried.

[0050] As is common in the art, the lyoprotectant is selected from one or a mixture of any proportion of trehalose, sucrose, lactose, glucose, raffinose, dextran, mannitol, sorbitol or xylitol, and the amount of the added lyoprotectant is 0.25-0.5%.

[0051] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A recombinant bovine interferon fusion protein, characterized in that: The fusion protein includes a key site for the antiviral effect of BoIFNα and a key site for the low toxicity effect of BoIFNτ, and the two are connected by a flexible linker; The key part of the antiviral effect of BoIFNα is the first 163 amino acids at the amino terminus of the BoIFNα mature peptide, and its amino acid sequence is shown in SEQ ID NO.1; The key site for the low toxicity of BoIFNτ is the 11 amino acids after the carboxyl end of the mature peptide of BoIFNτ, and its amino acid sequence is shown in SEQ ID NO.2; The flexible linker is GGSGGGS.

2. A variant protein of a recombinant bovine interferon fusion protein, wherein the variant protein is a protein obtained by mutating several specific amino acids in the recombinant bovine interferon fusion protein described in claim 1 into polar amino acids, wherein the above mutation improves the flexibility of the protein, while improving the isoelectric point and stability of the protein, thereby better maintaining the biological function of the protein; the variant protein is a protein obtained by mutating the 10th, 37th, and 119th amino acid residues (A10, A37, and A119, respectively) in BoIFNα and the 3rd and 6th amino acid residues from the bottom (M188 and D191, respectively) in BoIFNτ.

3. A variant protein of a recombinant bovine interferon fusion protein as claimed in claim 2, wherein the mutation is to mutate one or more of the specific amino acids into arginine, lysine or histidine.

4. The variant protein according to claim 3, characterized in that The amino acid sequence of the variant protein is shown in one of SEQ ID NO.4-10.

5. Use of the fusion protein according to claim 1 or the variant protein according to any one of claims 2 to 4 in the preparation of antiviral drugs for cattle.

6. A recombinant bovine long-acting interferon, characterized in that The recombinant bovine long-acting interferon is prepared by mixing the fusion protein according to claim 1 or the variant protein according to any one of claims 2 to 4 with a lyophilization protectant and then freeze-drying.

7. The recombinant bovine long-acting interferon according to claim 6, wherein the lyoprotectant is selected from one or a mixture of any proportion of trehalose, sucrose, lactose, glucose, raffinose, dextran, mannitol, sorbitol or xylitol, and the amount of the added lyoprotectant is 0.25-0.5%.

Images