CN113980142A

CN113980142A - Recombinant bovine interferon fusion protein and application thereof

Info

Publication number: CN113980142A
Application number: CN202111281067.3A
Authority: CN
Inventors: 许娜; 王燕; 石晶
Original assignee: Changchun Firefly Biotechnology Co ltd
Current assignee: Changchun Firefly Biotechnology Co ltd
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2022-01-28
Anticipated expiration: 2041-11-01
Also published as: CN113980142B

Abstract

The invention provides a recombinant bovine interferon fusion protein, which comprises bovine interferon alpha and a ricin B chain, wherein the bovine interferon alpha is bovine interferon mutant protein mBoIFN alpha, the ricin B chain is truncated ricin B chain protein RTBD1, and the mBoIFN alpha is a protein obtained by converting Seq ID NO: 1 to a polar amino acid, preferably by mutating one or several amino acids of the amino acid sequence as set forth in Seq ID NO: 1 to arginine or histidine at the 32 th and/or 83 th amino acid of the amino acid sequence shown in the specification. Compared with common bovine interferon alpha, the bovine interferon recombinant fusion protein provided by the invention has long half-life period and high biological activity, reduces the preparation cost of an interferon preparation, and has great potential and value in development of broad-spectrum antiviral drugs and veterinary clinical application.

Description

Recombinant bovine interferon fusion protein and application thereof

Technical Field

The invention belongs to the technical field of biological genetic engineering, and particularly relates to bovine interferon alpha recombinant protein and a preparation method thereof.

Background

Interferon (IFN) is a glycoprotein produced by host cells through an antiviral response reaction when an organism is infected with a virus, and has a prominent advantage in antiviral replication activity, and can enhance the activities of natural killer cells (NK cells), macrophages and T lymphocytes, regulate the immune function of the host, and exert its antiviral effect. IFNs are classified into types i, ii and iii according to the source, physicochemical properties, biological activity and recognition receptor of the interferon. The type I interferon can also be divided into IFN-alpha, IFN-beta and IFN-alpha, and the type I interferon has stronger antiviral activity.

At present, the application and development of I-type interferon cytokine medicaments for large mammals are slow. The existing bovine interferon mostly belongs to a single alpha type or a single beta type, but has the problems of short half-life period, easy degradation in vivo and the like in clinic, and has great significance in developing a safe and effective bovine IFN-alpha fusion protein preparation.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a recombinant bovine interferon fusion protein, which comprises bovine interferon alpha and ricin B chain, wherein the bovine interferon alpha is bovine interferon mutant protein mBoIFN alpha, the ricin B chain is truncated ricin B chain protein RTBD1, and the mBoIFN alpha is a protein obtained by converting Seq ID NO: 1 into polar amino acid. Respectively carrying out flexibility analysis on the wild type and the mutant protein, and if the flexibility of the mutant region protein is improved, mutating one or more amino acids in the region as mutation sites into polar amino acids.

The polar amino acid has hydrophilicity, improves the isoelectric point and the stability of the protein, changes the disulfide bond of the natural protein by mutating the selected site, and ensures that the prepared mutant protein is not easy to generate precipitate in the preservation process. Compared with wild protein, the three-dimensional structure of the mutant site selected by the invention has no change, thereby maintaining the biological function of the protein.

Preferably, the mBoIFN α is a peptide derived from Seq ID NO: 1, and the 32 th and/or 83 th amino acid of the amino acid sequence shown in the figure is mutated into polar amino acid.

Preferably, in any of the above, the polar amino acid comprises at least one of arginine or histidine.

Preferably according to any of the above, wherein the amino acid sequence of RTBD1 is Seq ID NO: 2, wherein n is 1, 2, 3 or 4, and m is more than or equal to 134.

Ricin B chain (RTB) is not toxic and has the function of enhancing the immune response of the body. The invention further preferably forms the fusion protein of the bovine IFN alpha and the RTBD1 truncated peptide to obtain the long-acting bovine interferon, can enhance the antiviral activity of the bovine interferon, and improve the protein stability of the interferon in the blood of a human body, thereby improving the half-life period of the interferon in the human body. Preferably, the amino acid sequence of said ricin B chain protein comprises Seq ID NO: 2, preferably, a truncated ricin B chain protein is used, the truncation comprising the addition of Seq ID NO: 2, 1 st amino acid, 1, 2 nd amino acid and 1 to 3 rd amino acid of the amino acid sequence shown in the sequence table; and/or converting Seq ID NO: 2, amino acid sequence knockout at 135 th to 263 th positions, amino acid sequence knockout at 136 th to 263 th positions, amino acid sequence knockout at 137 th to 263 th positions, amino acid sequence knockout at 138 th to 263 th positions, amino acid sequence knockout at 139 th to 263 th positions, amino acid sequence knockout at 140 th to 263 th positions, … …, amino acid sequence knockout at 260 th to 263 th positions, amino acid sequence knockout at 261 th to 263 th positions, amino acid sequence knockout at 262 th to 263 th positions, and amino acid sequence knockout at 263 th positions. The B chain truncated peptide of the ricin preferred by the invention reduces the size of protein molecules and increases the permeability of tissues in vivo.

Preferably any one of the above, truncated ricin B protein is RTBD1, the amino acid sequence of RTBD1 such as Seq ID NO: 18, respectively.

Preferably according to any of the above, the fusion protein comprises a peptide such as Seq ID NO: 3.

The invention also provides a gene encoding said fusion protein, said gene sequence comprising the amino acid sequence as set forth in Seq ID NO: 4.

The invention also provides a recombinant vector containing the gene.

The invention also provides a genetic engineering bacterium containing the recombinant vector, and the genetic engineering bacterium is obtained by transforming the recombinant vector into an escherichia coli host cell.

The invention also provides a method for preparing the fusion protein, which comprises the following steps: step one, constructing an mBoIFN alpha/RTBD 1 mutant vector; constructing an escherichia coli recombinant expression vector; step three, expressing the recombinant mBoIFN alpha/RTBD 1 fusion protein; step four, purification and renaturation.

The invention also provides application of the fusion protein in antiviral drugs. Application in preparing bovine antiviral drugs.

The invention provides a bovine interferon recombinant fusion protein and a preparation method and application thereof.

The preferred technical scheme of the invention is as follows:

the recombinant fusion protein of the invention is formed by connecting bovine interferon alpha and ricin B chain protein through a flexible linker. Preferred bovine interferon alpha is bovine IFN alpha mutant protein mbboifn alpha, with an amino acid sequence such as Seq ID NO: shown at 17. A preferred ricin B chain protein is a truncated ricin B chain RTBD1, amino acid sequence such as Seq ID NO: 18, respectively. The recombinant fusion protein containing the bovine IFN alpha mutant protein and RTBD1 is named mBoIFN alpha/RTBD 1. Preferably, the flexible linker is a (Gly4Ser)3 linking peptide.

The invention provides a preparation method of bovine interferon recombinant fusion protein, which mainly comprises the following steps:

step one, constructing an mBoIFN alpha/RTBD 1 mutant vector;

constructing an escherichia coli recombinant expression vector;

step three, expressing the recombinant mBoIFN alpha/RTBD 1 fusion protein;

step four, purification and renaturation.

As a preferred embodiment, the step one specifically comprises the following steps:

(1) bioinformatics analysis of potential mutation sites for BoIFN α: carrying out homologous modeling on bovine interferon alpha and a bovine I-type interferon receptor through an online public database, obtaining a molecular docking structure model by using Z-DOCK, carrying out alanine scanning mutation on a full-length sequence of the I-type interferon, analyzing the change of the binding force between the interferon and the receptor before and after mutation, and screening to obtain potential mutation sites (the selection principle of the mutation sites is that the change of the binding force between IFN and the IFN receptor before and after the mutation is predicted and evaluated, a key site for maintaining the three-dimensional structure of the protein is calculated, then, a plurality of non-key sites are used as candidates of the mutation sites to carry out site-directed mutation on different natural amino acids, and a site and an amino acid with higher binding force in a final mutation result are selected);

(2) construction of a cloning vector for a BoIFN alpha mutant gene: bovine IFN alpha site-directed mutagenesis primers (the name and sequence of the primers are as follows: F: Seq ID NO: 5; R: Seq ID NO: 6; PM1F: Seq ID NO: 7; PM1R: Seq ID NO: 8; PM2F: Seq ID NO: 9; PM2R: Seq ID NO: 10) were designed, pMD18T-BoIFN alpha constructed in the laboratory was used as a template (constructing a bovine interferon IFN alpha (BoIFN alpha) nucleotide sequence shown in Seq ID NO: 19 into a pMD18T vector to obtain pMD 18T-IFN alpha is a conventional technique in the art and will not be described herein in detail), amino acids 32 and 83 in the bovine IFN alpha sequence are mutated to arginine, the binding ability of bovine IFN alpha to a type 1 interferon receptor is increased to enhance the antiviral activity, and the mutated sequence is named as mBoIFN alpha. The mutated mBoIFN alpha is connected to a pMD18T vector, and the product with correct sequencing is named as: pMD18T-mBoIFN alpha plasmid;

(3) construction of RTB truncated gene cloning vector: RTBD1 truncated primers (the names and sequences of the primers are shown as follows: Seq ID NO: 11 and Seq ID NO: 12) were designed, pMD18T-RTB constructed in the laboratory was used as a template (constructing the nucleotide sequence of the full-length RTB shown in Seq ID NO: 20 into pMD18T vector to obtain pMD18T-RTB is a conventional technique in the art and is not described herein in detail), the 10 th to 402 th nucleotide sequences were called from the full-length RTB sequence, corresponding to the 4 th methionine to 134 th threonine in the amino acid sequence of RTB, and the called gene was named RTBD 1. The obtained gene is connected to a pMD18T cloning vector, and a product with correct sequencing is named as a pMD18T-RTBD1 plasmid;

(4) construction of mBoIFN alpha/RTBD 1 fusion Gene cloning vector: overlapping PCR primers (primer name and sequence are as follows: F: Seq ID NO: 13; Seq ID NO: 14, Seq ID NO: 15, Seq ID NO: 16) containing (G4S)3linker gene were designed to construct correct cloned plasmids pMD18T-mBoIFN α and pMD18T-RTBD1 as templates, and a fusion gene composed correctly as 5 '-mBoIFN α -linker-RTBD 1-3' was amplified and named mBoIFN α/RTBD 1. The fusion gene was ligated into the pMD18T vector and the correctly sequenced product was named pMD18T-mBoIFN α/RTBD1 plasmid for downstream genetic engineering.

As a preferred embodiment, the second step specifically comprises the following steps:

carrying out double enzyme digestion on the pMD18T-mBoIFN alpha/RTBD 1 plasmid and the pET28a vector with correct sequencing respectively, wherein the reaction system and conditions are shown in Table 1, and carrying out gel recovery on double enzyme digestion products; the two products after the glue recovery are connected by using T4 ligase, and the connection system and conditions are shown in the following table 2;

TABLE 1 recombinant plasmid double digestion System

TABLE 2 recombinant plasmid ligation System

Transforming the ligation product into a Trans10 competent cell; after transformation, coating the obtained product on an LB solid plate with the final concentration of 100 mu g/mL Kan + resistance, after overnight culture, selecting a plurality of white single colonies with good growth vigor, carrying out amplification culture and carrying out PCR; the positive plasmids identified by PCR were subjected to sequencing analysis.

As a preferred embodiment, the step three specifically includes the following steps:

inoculating correctly sequenced positive bacteria BL21(DE3)/PET28a-mBoIFN alpha/RTBD 1 into 5mL LB culture medium containing Kan with the concentration of 100 mu g/mL according to the proportion of 1:100v/v, carrying out constant temperature shaking culture at 180rpm at 37 ℃ until OD600 is 0.6, then transferring the positive bacteria to 500mL of the same LB culture medium according to the proportion of 1:100v/v, supplementing Kan until the final concentration is 100 mu g/mL, and carrying out constant temperature shaking culture at 180rpm at 37 ℃ until OD600 is 0.6; adding IPTG to the induction group until the final concentration is 1mmol/L, and oscillating at constant temperature of 180rpm at 37 ℃ for induction for 16 h; after 16h of induction, the bacterial pellets were obtained by centrifugation at 8000rpm and 4 ℃.

As a preferred embodiment, the step four specifically includes the following steps:

(1) ion exchange chromatography;

(2) metal chelating chromatography;

(3) rmBoIFN α/RTBD1 protein renaturation.

As a preferred embodiment, the step (1) specifically includes the steps of:

a protein purification A liquid balance Q column (strong anion exchange column) is used, rmBoIFN alpha/RTBD 1 inclusion body dissolving liquid is loaded on the column, and flow-through liquid is collected, wherein the target protein mainly exists in the flow-through liquid. The salt ion concentration of the flow-through was adjusted to 0.5mol/L for the next step of metal chelate chromatography.

As a preferred embodiment, the step (2) specifically includes the steps of:

the Ni2+ chemical Sepharose loading was equilibrated with protein purification B solution; loading the rmBoIFN alpha/RTBD 1 solution purified by ion exchange chromatography to a column, mixing the protein purification solution B and the protein purification solution C by a gradient mixing module in a protein purification system, eluting chemical Sepharose by imidazole with final concentration of 50mmol/L and 250mmol/L respectively, and collecting protein flow-through solution, hybrid protein solution and target protein solution respectively; the protein solution collected at each step was subjected to SDS-PAGE analysis at a gel concentration of 12%. (gradient mixing module in protein purification system, module in Akta protein purification system, instrument manufacturer of American general company, model Akta explorer.)

As a preferred embodiment, the step (3) specifically includes the steps of:

diluting the rmBoIFN alpha/RTBD 1 protein solution purified by two steps with protein renaturation A solution until the concentration of rmBoIFN alpha/RTBD 1 is 0.1mg/mL, filling the protein renaturation A solution into a protein ultrafiltration system with the molecular weight cutoff of 3000 for dialysis renaturation, and adding protein renaturation B solution into the protein ultrafiltration system; regulating the rotating speed of a peristaltic pump in the protein ultrafiltration system and the tightness degree of a liquid outlet valve to keep the protein concentration speed consistent with the liquid inlet speed of the liquid B; the protein renaturation process is carried out at 4 ℃, and the whole process is carried out for 48-72 h; and after the protein renaturation B liquid flows out, introducing the protein renaturation C liquid to remove residual cane sugar and glycerin components in the renaturation process, concentrating rmBoIFN alpha/RTBD 1 protein until the concentration is 1-1.5mg/mL, and obtaining the recombinant mBoIFN alpha/RTBD 1 fusion protein.

The bovine interferon recombinant fusion protein can be applied to preparation of antiviral drugs.

The invention has the beneficial effects that: the bovine interferon recombinant fusion protein is formed by connecting bovine interferon alpha and ricin B chain protein through a flexible linker, wherein the flexible linker selects (Gly4Ser)3 connecting peptide. Compared with the common bovine interferon alpha, the bovine interferon recombinant fusion protein has long half-life period and high biological activity, prolongs the half-life period, improves the biological activity, reduces the preparation cost of an interferon preparation, and has great potential and value in development of broad-spectrum antiviral drugs and veterinary clinical application.

Drawings

FIG. 1 comparison of three-dimensional Structure of bovine IFN α mutein and wild-type bovine IFN α protein in preferred embodiment 2 of the present invention

Detailed Description

The present invention will be more clearly and completely described in the following embodiments, but the described embodiments are only a part of the embodiments of the present invention, and not all of them. The examples are provided to aid understanding of the present invention and should not be construed to limit the scope of the present invention.

Example 1 preparation of recombinant mBoIFN alpha/RTBD 1 fusion protein

1. Construction of mBoIFN alpha/RTBD 1 mutant vector

(1) Bioinformatics analysis of potential mutation sites for BoIFN α: carrying out homologous modeling on bovine interferon alpha and a bovine I-type interferon receptor through an online public database, obtaining a molecular docking structure model by using Z-DOCK, carrying out alanine scanning mutation on a full-length sequence of the I-type interferon, analyzing the binding force change of the interferon and the receptor before and after mutation, and screening to obtain a potential mutation site;

(2) construction of a cloning vector for a BoIFN alpha mutant gene: designing a bovine IFN alpha site-directed mutation primer, taking pMD18T-BoIFN alpha constructed in a laboratory as a template, mutating the amino acid aspartic acid at the 32 th site and the amino acid aspartic acid at the 83 th site in a bovine IFN alpha sequence into arginine, increasing the binding capacity of the bovine IFN alpha and a type 1 interferon receptor so as to enhance the antiviral activity of the bovine IFN alpha, and naming the mutated sequence as mBoIFN alpha. The mutated mBoIFN alpha is connected to a pMD18T vector, and the product with correct sequencing is named as: pMD18T-mBoIFN alpha plasmid;

(3) construction of RTB truncated gene cloning vector: designing a truncated primer of RTBD1, taking pMD18T-RTB constructed in a laboratory as a template, and calling the 10 th to 402 th nucleotide sequences from the full-length RTB sequence, wherein the calling gene is named as RTBD1 corresponding to the 4 th methionine to 134 th threonine from the RTB amino acid sequence. The obtained gene is connected to a pMD18T cloning vector, and a product with correct sequencing is named as a pMD18T-RTBD1 plasmid;

(4) construction of mBoIFN alpha/RTBD 1 fusion Gene cloning vector: an overlapping PCR primer containing (G4S)3linker gene is designed to construct correct cloning plasmid pMD18T-mBoIFN alpha and pMD18T-RTBD1 as templates, and a fusion gene which takes 5 '-mBoIFN alpha-linker-RTBD 1-3' as a correct composition is obtained by amplification, and is named mBoIFN alpha/RTBD 1. The fusion gene was ligated into the pMD18T vector and the correctly sequenced product was named pMD18T-mBoIFN α/RTBD1 plasmid for downstream genetic engineering. 2. Construction of recombinant expression vector for Escherichia coli

The pMD18T-BoIFN alpha/RTB plasmid and the pET28a vector which are correctly sequenced are subjected to double enzyme digestion respectively, the reaction system and conditions are shown in Table 1, and the double enzyme digestion products are subjected to gel recovery. The two products after gel recovery were ligated by T4 ligase, and the ligation system and conditions are shown in table 2.

The ligation product was transformed into Trans10 competent cells. After transformation, the cells were spread on Kan + -resistant LB solid plates with a final concentration of 100. mu.g/mL, cultured overnight, and then a plurality of white single colonies with good growth were picked, subjected to amplification culture, and subjected to PCR. And (3) sending the positive plasmid which is correctly identified by the PCR to Shanghai biological engineering technical service company Limited for sequencing analysis.

3. Recombinant mBoIFN alpha/RTBD 1 fusion protein expression

Inoculating the positive bacterium BL21(DE3)/PET28a-mBoIFN alpha/RTBD 1 with correct sequencing in 5mL LB culture medium containing 100 mug/mL Kan according to the ratio of 1:100v/v, carrying out shaking culture at constant temperature of 180r/min at 37 ℃ until OD600 is 0.6, transferring the positive bacterium to 500mL of the same LB culture medium according to the ratio of 1:100v/v, supplementing Kan until the final concentration is 100 mug/mL, and carrying out shaking culture at constant temperature of 180r/min at 37 ℃ until OD600 is 0.6; adding IPTG to the induction group until the final concentration is 1mmol/L, and oscillating and inducing at constant temperature of 180r/min for 16h at 37 ℃; after 16h of induction, the bacterial pellet is obtained by centrifugation at 8000r/min and 4 ℃.

4. Fusion protein purification and renaturation

The pellet of rmBoIFN α/RTBD1 was purified using a two-step purification procedure. The purification method is carried out in the order of ion exchange chromatography and metal chelating chromatography, and comprises the following steps:

(1) ion exchange chromatography: a Q column was equilibrated with protein purification A solution (50mmol/L PB, 8mol/L Urea, pH 6.0), inclusion bodies of rmBoIFN α/RTBD1 were denatured with protein purification A solution and the lysate was applied to the column, and the flow-through solution, in which the protein of interest was mainly present, was collected. The salt ion concentration of the flow-through was adjusted to 0.5mol/L for the next step of metal chelate chromatography.

(2) Metal chelating chromatography: the loading of Ni2+ chemical Sepharose was equilibrated with protein purification B solution (50mmol/L PB, 8mol/L Urea, 0.5mol/L NaCl pH 6.0). Putting the rmBoIFN alpha/RTBD 1 solution purified by ion exchange chromatography on a column, mixing a protein purification solution B and a protein purification solution C (50mmol/L Tris, 8mol/L Urea, 0.5mol/L NaCl, 500mmol/L Imidazole pH 8.5) by a gradient mixing module in a protein purification system, eluting chemical Sepharose by using Imidazole with final concentration of 50mmol/L and 250mmol/L respectively, and collecting a protein flow-through solution, a protein mixed solution and a target protein solution respectively. The protein solution collected at each step was subjected to SDS-PAGE analysis at a gel concentration of 12%.

(3) rmBoIFN α/RTBD1 protein renaturation: the two-step purified rmBoIFN α/RTBD1 protein solution was diluted with protein renaturation A solution (50mmol/L Tris, 8mol/L Urea, 10% m/v sucrose, 10% v/v glycerol, 5mmol/L DTT, pH 8.5) to a concentration of 0.1mg/mL rmBoIFN α/RTBD1 and filled into a protein ultrafiltration system with a molecular weight cutoff of 3000 for dialytic renaturation, and protein renaturation B solution (50mmol/L Tris, 10% m/v sucrose, 10% v/v glycerol, pH 8.5) was added to the protein ultrafiltration system. The rotating speed of a peristaltic pump in the protein ultrafiltration system and the tightness degree of a liquid outlet valve are adjusted, so that the protein concentration speed is consistent with the liquid inlet speed of the liquid B. The protein renaturation process was carried out at 4 ℃ and the whole procedure was carried out for 48-72h to satisfy the complete removal of urea and the slow and sufficient folding of rmBoIFN α/RTBD1 protein. And after the protein renaturation B liquid flows out, introducing a protein renaturation C liquid (50mmol/L Tris, pH 8.5) to remove components such as sucrose, glycerol and the like remained in the renaturation process, concentrating rmBoIFN alpha/RTBD 1 protein until the concentration is 1-1.5mg/mL, and obtaining the recombinant mBoIFN alpha/RTBD 1 fusion protein.

Example 2 three-dimensional Structure verification of bovine IFN alpha muteins

A bovine IFN alpha mutein (mBoIFN alpha, protein sequence shown in Seq ID NO: 17) was prepared in the same manner as the fusion protein of example 1. The obtained bovine IFN alpha mutein has NO change in three-dimensional structure as compared with the wild type bovine IFN alpha protein (protein sequence is shown in Seq ID NO: 1), as shown in FIG. 1.

Example 3 in vitro antiviral Activity assay of recombinant mBoIFN alpha/RTBD 1 fusion proteins

Bovine kidney cells (MDBK) were treated with 10-fold dilutions of rmBoIFN α/RTBD1 protein for 24h, followed by 100 XTCID₅₀Bovine Vesicular Stomatitis Virus (VSV) infection at concentrations to suppress 50% of cytopathic interferon maximumThe dilution is determined as 1 interferon activity unit, and the detection result shows that the recombinant fusion protein shows very high VSV inhibiting activity and the virus titer reaches 3.84 x 10⁷U/mL, the antiviral specific activity of the recombinant fusion protein is 3.2 multiplied by 10⁷U/mg。

Example 4 detection of immunomodulatory Activity of recombinant mBoIFN alpha/RTBD 1 fusion proteins

Six cattle with approximately same weight are divided into two groups, which are marked as experimental group and control group, and the neck part of the experimental group is injected with 3 multiplied by 10 subcutaneously⁶U recombinant mBoIFN alpha/RTBD 1, control group neck subcutaneous injection equal volume saline, injection after 1 week cattle peripheral blood, then weekly blood, using lymphocyte separation liquid to separate lymphocytes, using serum-free cell culture medium washing 3 times, using complete culture medium heavy suspension, adjusting cell concentration to 2 x 10⁶Per mL, 24-well cell culture plates 1mL of lymphocytes per well, 37 ℃ 5% CO₂Culturing for 72h under the condition, and collecting cell culture solution supernatant. The ELISA method detects the content of IL4 and IFN gamma in the culture supernatant, and the detection result is shown in Table 3:

TABLE 3 bovine cellular immune response levels in each group

Example 5 pharmacokinetic Studies of recombinant mBoIFN α/RTBD1 fusion proteins

Measuring half-life of rmBoIFN α/RTBD1 by determining the relationship between blood concentration and time of rmBoIFN α/RTBD1 by cytopathy inhibition;

taking 6 female cattle, dividing into 2 groups (3 cattle/group): respectively carrying out intramuscular injection on 2mg/mL rmBoIFN alpha/RTBD 1 and 0.5mL BoIFN alpha on a long-acting interferon group and a common interferon group, carrying out intravenous blood sampling for 1h, 2h, 4h, 8h, 16h, 24h, 48h and 72h after injection, standing blood at the low temperature of 4 ℃, and centrifuging at the low temperature of 3000r/min for 5min to obtain upper serum;

the long-acting interferon group is recombinant fusion protein of rmBoIFN alpha/RTBD 1 provided by the invention as recombinant bovine long-acting interferon, and the amino acid sequence of the common interferon (or called natural interferon) protein used by the invention is shown as Seq ID NO: 1 is shown.

Determining the rmBoIFN alpha/RTBD 1 concentration in serum at different time points by adopting a cytopathic effect inhibition method, performing curve fitting by using DAS pharmacokinetic software and calculating related parameters;

the half-life of each group was determined as shown in table 4 below, and the half-life of rmBoIFN α/RTBD1 was much higher than BoIFN α, enabling a significant increase in half-life.

TABLE 5 determination of half-life of drugs

Compared with the half-life of natural interferon about 3.1h, the half-life of rmBoIFN alpha/RTBD 1 is greatly improved, and the half-life is improved to 2.77 times.

The invention discloses a bovine interferon recombinant fusion protein, a preparation method and application thereof, and a person skilled in the art can refer to the content and appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the products described herein without departing from the spirit and scope of the invention.

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ctgaggaggg tctccccttc ctcctgcctg cagcgcagaa atgacttcgc attcccccag 120

gaggcgctgg gtggcagcca gttgcagaag gctcaagcca tctctgtgct ccacgaggtg 180

acccaacaca ccttccagct tttcagcaca gagggctcgg ccgctgcgtg ggatgagagc 240

ctcctgcgca agctccgagc tgcactggat cagcagctca ctgacctgca agcctgtctg 300

aggcaggagg aggggctgcg aggggctccc ctgctcaagg aggatgccag cctggctgtg 360

aggaaatact tccacagact cactctctat ctgcgagaga agagacacaa cccttgtgcc 420

tgggaggttg tcagagcaga agtcatgaga gccttctctt cctcaacaaa cttgcaggag 480

agattcagga gaaaggacgg tggcggcggt agtggcggcg gtggcagcgg tggtggtggt 540

tcaatggacc ctgaaccgat tgttcgcatt gttggtcgca atggcctgtg cgtggatgtg 600

cgcgatggtc gttttcataa tggtaatgcc attcagctgt ggccgtgtaa aagcaatacc 660

gatgcaaatc agctgtggac cctgaaacgt gataatacca ttcgtagcaa tggcaaatgc 720

ctgaccacct atggctatag tccgggtgtt tatgtgatga tctatgattg taataccgca 780

gccaccgatg ccacccgctg gcagatttgg gataatggta caattattaa cccgcgcagt 840

agtctggtgc tggcagcaac cagcggcaat agtggtacaa ccctgaccgt tcagaccaat 900

atctatgccg ttagccaggg ttggctgccg acc 933

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

catatgtgcc acctgcctca ca 22

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtcctttctc ctgaatct 18

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cctgcagcgc agaaatgac 19

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aagtcatttc tgcgctgca 19

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gagcctcctg cgcaagct 18

<210> 10

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcggagcttg cgcagga 17

<210> 11

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atggatccgg aaccgata 18

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gaattctaag gtcggcagcc a 21

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

catatgtgcc acctgcctca ca 22

<210> 14

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggtggcggcg gtagtggcgg cggtggcagc ggtggtggtg gttcaatgga ccctgaaccg 60

<210> 15

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tgaaccacca ccaccgctgc caccgccgcc actaccgccg ccaccgtcct ttctcctg 58

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gaattcaatg gtcggcagcc aacc 24

<210> 17

<211> 166

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Cys His Leu Pro His Thr His Ser Leu Pro Asn Arg Arg Val Leu Thr

1 5 10 15

Leu Leu Arg Gln Leu Arg Arg Val Ser Pro Ser Ser Cys Leu Gln Arg

20 25 30

Arg Asn Asp Phe Ala Phe Pro Gln Glu Ala Leu Gly Gly Ser Gln Leu

35 40 45

Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Val Thr Gln His Thr

50 55 60

Phe Gln Leu Phe Ser Thr Glu Gly Ser Ala Ala Ala Trp Asp Glu Ser

65 70 75 80

Leu Leu Arg Lys Leu Arg Ala Ala Leu Asp Gln Gln Leu Thr Asp Leu

85 90 95

Gln Ala Cys Leu Arg Gln Glu Glu Gly Leu Arg Gly Ala Pro Leu Leu

100 105 110

Lys Glu Asp Ala Ser Leu Ala Val Arg Lys Tyr Phe His Arg Leu Thr

115 120 125

Leu Tyr Leu Arg Glu Lys Arg His Asn Pro Cys Ala Trp Glu Val Val

130 135 140

Arg Ala Glu Val Met Arg Ala Phe Ser Ser Ser Thr Asn Leu Gln Glu

145 150 155 160

Arg Phe Arg Arg Lys Asp

165

<210> 18

<211> 130

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Met Asp Pro Glu Pro Ile Val Arg Ile Val Gly Arg Asn Gly Leu Cys

1 5 10 15

Val Asp Val Arg Asp Gly Arg Phe His Asn Gly Asn Ala Ile Gln Leu

20 25 30

Trp Pro Cys Lys Ser Asn Thr Asp Ala Asn Gln Leu Trp Thr Leu Lys

35 40 45

Arg Asp Asn Thr Ile Arg Ser Asn Gly Lys Cys Leu Thr Thr Tyr Gly

50 55 60

Tyr Ser Pro Gly Val Tyr Val Met Ile Tyr Asp Cys Asn Thr Ala Ala

65 70 75 80

Thr Asp Ala Thr Arg Trp Gln Ile Trp Asp Asn Gly Thr Ile Ile Asn

85 90 95

Pro Arg Ser Ser Leu Val Leu Ala Ala Thr Ser Gly Asn Ser Gly Thr

100 105 110

Thr Leu Thr Val Gln Thr Asn Ile Tyr Ala Val Ser Gln Gly Trp Leu

115 120 125

Pro Thr

130

<210> 19

<211> 498

<212> DNA

<213> cattle (Bos taurus)

<400> 19

tgccacctgc ctcacaccca cagcctgccc aacaggaggg tcctgacact cctgcgacaa 60

ctgaggaggg tctccccttc ctcctgcctg caggacagaa atgacttcgc attcccccag 120

gaggcgctgg gtggcagcca gttgcagaag gctcaagcca tctctgtgct ccacgaggtg 180

acccaacaca ccttccagct tttcagcaca gagggctcgg ccgctgcgtg ggatgagagc 240

ctcctggaca agctccgagc tgcactggat cagcagctca ctgacctgca agcctgtctg 300

aggcaggagg aggggctgcg aggggctccc ctgctcaagg aggatgccag cctggctgtg 360

aggaaatact tccacagact cactctctat ctgcgagaga agagacacaa cccttgtgcc 420

tgggaggttg tcagagcaga agtcatgaga gccttctctt cctcaacaaa cttgcaggag 480

agattcagga gaaaggac 498

<210> 20

<211> 798

<212> DNA

<213> Castor (Ricinus communis)

<400> 20

gcagatgttt gcatggaccc tgaaccgatt gttcgcattg ttggtcgcaa tggcctgtgc 60

gtggatgtgc gcgatggtcg ttttcataat ggtaatgcca ttcagctgtg gccgtgtaaa 120

agcaataccg atgcaaatca gctgtggacc ctgaaacgtg ataataccat tcgtagcaat 180

ggcaaatgcc tgaccaccta tggctatagt ccgggtgttt atgtgatgat ctatgattgt 240

aataccgcag ccaccgatgc cacccgctgg cagatttggg ataatggtac aattattaac 300

ccgcgcagta gtctggtgct ggcagcaacc agcggcaata gtggtacaac cctgaccgtt 360

cagaccaata tctatgccgt tagccagggt tggctgccga ccaataatac ccagccgttt 420

gttaccacca ttgttggtct gtatggcatg tgtctgcaag ccaatagtgg taaagtgtgg 480

ctggaagatt gtacaagcga aaaagccgaa cagcagtggg ccctgtatgc cgatggtagc 540

attcgcccgc agcagaatcg cgatagttgc ctgaccacag atgccaatat taaaggcacc 600

gttgttaaaa ttctgagctg cggcccggcc agtagcggcc aacgttagat gtttaaaaat 660

gatggcacca ttctgaatct gtatattggc ctggttctgg atgtgcgtcg cagcgatccg 720

agcagtctga aacagattat tgttcatccg gttcatggca atctgaatca gatttggtta 780

ccgctgtttt aagaattc 798

Claims

1. A recombinant bovine interferon fusion protein comprising bovine interferon alpha and ricin B chain, wherein said bovine interferon alpha is bovine interferon mutant protein mbboifn alpha, and said ricin B chain is truncated ricin B chain protein RTBD1, said mbboifn alpha is a polypeptide that converts Seq ID NO: 1 into polar amino acid.

2. The fusion protein of claim 1, wherein the mBoIFN α is a fusion protein of Seq ID NO: 1, and the 32 th and/or 83 th amino acid of the amino acid sequence shown in the figure is mutated into polar amino acid.

3. The fusion protein of claim 2, wherein the polar amino acid comprises at least one of arginine or histidine.

4. The fusion protein of any one of claims 1-3, wherein the amino acid sequence of RTBD1 is Seq ID NO: 2, wherein n is 1, 2, 3 or 4, and m is more than or equal to 134.

5. The fusion protein of claim 4, wherein the fusion protein comprises an amino acid sequence as set forth in Seq ID NO: 3.

6. A gene encoding the fusion protein of claim 5, wherein the gene sequence comprises the sequence set forth in Seq ID NO: 4.

7. A recombinant vector comprising the gene of claim 6.

8. A genetically engineered bacterium containing the recombinant vector of claim 7, wherein the genetically engineered bacterium is obtained by transforming the recombinant vector of claim 7 into an Escherichia coli host cell.

9. A method for preparing the fusion protein of any one of claims 1 to 5, comprising the steps of: step one, constructing an mBoIFN alpha/RTBD 1 mutant vector; constructing an escherichia coli recombinant expression vector; step three, expressing the recombinant mBoIFN alpha/RTBD 1 fusion protein; step four, purification and renaturation.

10. Use of the fusion protein of any one of claims 1 to 5 in an antiviral medicament.