CN110904115B

CN110904115B - Canine recombinant interferon alpha 7, preparation method and application thereof, expression vector containing canine recombinant interferon alpha 7 and host cell

Info

Publication number: CN110904115B
Application number: CN201911371317.5A
Authority: CN
Inventors: 王红朵; 刘洁; 李媛媛; 杨欣怡; 何志远; 单雪芹; 许高涛; 周炜; 孔国庆; 金巢
Original assignee: Wuhu Yingtefeier Biological Products Industry Research Institute Co ltd; Anhui Jiuchuan Biotechnology Co ltd
Current assignee: Wuhu Yingtefeier Biological Products Industry Research Institute Co ltd; Anhui Jiuchuan Biotechnology Co ltd
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2021-08-17
Anticipated expiration: 2039-12-25
Also published as: CN110904115A

Abstract

The invention discloses a canine recombinant interferon alpha 7, a preparation method and application thereof, an expression vector and a host cell containing the canine recombinant interferon alpha 7, a nucleotide sequence of the canine recombinant interferon alpha 7 is obtained by codon optimization, a pichia pastoris strain expression system is utilized to express recombinant yeast engineering bacteria containing the codon optimized canine recombinant interferon alpha 7 gene of the invention, and the high-activity canine recombinant interferon alpha 7 with secretory expression is obtained⁶IU/mg, the research on the expression, fermentation and activity of CaIFN-alpha 7 in a eukaryotic yeast system has important significance for realizing the industrial production of the canine alpha interferon early.

Description

Canine recombinant interferon alpha 7, preparation method and application thereof, expression vector containing canine recombinant interferon alpha 7 and host cell

Technical Field

The invention belongs to the field of biological products, and discloses canine recombinant interferon alpha 7, a preparation method and application thereof, an expression vector containing the canine recombinant interferon alpha 7 and a host cell.

Background

In recent years, the pet industry in China is rapidly developed, the number of pet dogs and cats is increasing, and the pet-derived viral epidemic disease is a disease with serious harm at present. The most common canine distemper, canine parvovirus disease and the like are generally serious in harm, high in morbidity, strong in infectivity and high in mortality, are the most serious infectious diseases which harm the canine raising industry in China, and seriously restrict the development of the pet industry. Traditional treatment regimens are clinically poorly effective, and active treatment and prevention of canine viral diseases is therefore a major concern in the industry.

Interferons are important substances with comprehensive antiviral effect, enhanced specific and nonspecific immune response, and simultaneously capable of regulating the growth process of normal cells and tumor cells. Since the discovery of interferon, the existence of interferon has been found in various mammals, fishes, insects, and the like, confirming the wide existence of interferon. Based on the gene homology, structural features, receptor and biological activity of different interferons, the interferons are classified into 3 types, i.e. type I, type II and type III. Type I interferons include IFN-alpha, IFN-beta, IFN-delta, IFN-epsilon, IFN-kappa, IFN-tau and IFN-omega, type II includes only gamma interferon, and IFN-lambda is the type III interferon found in 2003.

The canine interferon-alpha gene has the full length of 564 nucleotides, encodes 187 amino acids and consists of a signal peptide with 23 amino acids and a mature protein with 164 amino acids. The CaIFN-alpha contains 6 cys and 2 potential N-glycosylation sites, and the amino acid sequence homology of CaIFN-alpha 4-8 and the previously reported subtype CaIFN-alpha 1-3 is 93-100%. CaIFN- α is divided into two groups based on the amino acid sequence and molecular phylogenetic tree: group I was CaIFN

alpha

1, 2 and 7, and group II was CaIFN-

alpha

3, 4, 5, 6 and 8.

At present, because the recombinant interferon expressed by pronucleus can not be subjected to posttranslational modification, the space conformation of the recombinant interferon has a certain difference with that of natural interferon, and the immunogenicity of the recombinant interferon is lower.

Disclosure of Invention

The invention aims to provide a canine recombinant interferon alpha 7, a preparation method and application thereof, an expression vector containing the canine recombinant interferon alpha 7 and a host cell, wherein on the basis of not changing the canine interferon alpha 7 amino acid sequence, codons are optimized, and the canine recombinant interferon alpha 7 which can be efficiently expressed and has high purity is prepared.

The technical scheme adopted by the invention is as follows:

the nucleotide sequence of the canine recombinant interferon alpha 7 is shown as SEQ ID No. 3.

The invention also provides a recombinant expression vector containing the nucleotide sequence of the canine recombinant interferon alpha 7.

Further, the expression vector is pPIC 9K-CaIFN-alpha 7. In order to simply and rapidly obtain high-expression transformants, the screening of high-copy clones of a target gene is often carried out, so that a resistance gene such as a G418 (geneticin) -resistant gene is introduced into the vectors, and then a pPIC9K expression vector is obtained, so that the high-copy expression clones can be screened by increasing the concentration of G418. pPIC9K contains the bacterial kan gene and confers geneticin resistance to Pichia pastoris. The level of geneticin resistance depends largely on the number of integrated kan genes. After a single copy of pPIC9K was integrated into the Pichia genome, the Pichia was conferred a level of geneticin resistance of about 0.25 mg/ml. Any vector multicopy integration can increase the level of geneticin resistance, from 0.5mg/ml (1-2 copies) to 4mg/ml (7-12 copies). Due to the genetic linkage between the kan gene and the expression cassette (pAOX1 and the target gene), it can be concluded from the high resistance to geneticin that the clone contains multiple copies of the gene of interest. Expression of the protein may be increased due to the dose benefit of the gene.

Since the pPIC9K expression vector can be used for screening high-copy transformants, but the expression vector does not carry a Tag which is convenient for purification, the HIS6-Tag is added at the N end of the amino acid when the gene is synthesized, and the protein of interest can be purified by affinity chromatography.

The invention also provides a host cell containing the nucleotide sequence of the canine recombinant interferon alpha 7 or the recombinant expression vector.

Furthermore, the host cell is a pichia pastoris cell strain GS115, the GS115 contains HIS4 gene mutation and cannot synthesize histidine, and the expression vector pPIC9K contains the HIS4 gene as a selection marker, so that a transformant can grow in a histidine-deficient culture medium, and the GS115 is selected as the expression host cell to facilitate the selection of the transformant.

Pichia pastoris, as unicellular organism, is easy for gene construction and culture in laboratories, and as eukaryote it has the ability to modify proteins post-translationally, such as hydrolysis, folding, disulfide bond formation and glycosylation of proteins, so many proteins which are inactive or have low activity after being expressed in prokaryotic expression system can be successfully expressed in Pichia pastoris expression system. Compared with other expression systems, the expression system has the characteristics of relatively quick expression, simple operation, low experiment and production cost, high economical efficiency and high yield.

The invention also provides a preparation method of the canine recombinant interferon alpha 7, which comprises the following steps:

(1) connecting the nucleotide sequence gene of the canine recombinant interferon alpha 7 with a vector pPIC9K, transforming the gene into an escherichia coli competent cell DH5a, culturing, selecting a single colony, extracting a plasmid, and performing PCR identification;

(2) selecting a bacterial colony which is identified by PCR and sequenced correctly for amplification culture, then extracting recombinant plasmids, linearizing the recombinant plasmids, and then purifying;

(3) electrically transforming the linearized recombinant plasmid obtained in the step (2) into a pichia pastoris cell strain GS115, and sequentially carrying out incubation and culture until the diameter of a bacterial colony is 1 mm;

(4) culturing the recombinant strain GS115/pPIC 9K-CaIFN-alpha 7 of the positive clone strain obtained in the step (3) in a BMGY culture medium until the OD600 value of the strain is 2-6, centrifuging, diluting the strain precipitate with an induction culture medium BMMY until the OD600 value is 1-2, culturing for 4-5 days, supplementing methanol every 24 hours until the final concentration is 1%, performing induction culture, and centrifuging to collect a supernatant; after purification, the canine recombinant interferon-alpha 7 protein can be obtained.

The invention provides application of the canine recombinant interferon alpha 7 in preparation of a VSV virus resistant medicament.

The canine recombinant interferon alpha 7 provided by the invention can be further prepared into a biological product which is further applied to antiviral drugs, and the biological product is preferably a freeze-dried preparation.

Compared with the prior art, the codon-optimized nucleotide sequence gene is directionally cloned into a pichia pastoris expression plasmid to construct a recombinant plasmid, the recombinant plasmid is converted into a pichia pastoris competent cell to prepare a recombinant bacterium, fermentation and expression of the yeast are optimized by optimizing methanol induction conditions, induction concentration, induction time, protein harvesting time and the like, and the recombinant bacterium is purified by adopting an affinity chromatography method, so that the yield of a product is effectively improved, and the canine recombinant interferon-alpha 7 with high yield, high purity and high activity is obtained, wherein the antiviral activity of the canine interferon alpha 7 recombinant protein is 1.03 multiplied by 10⁶IU/mL, specific activity 1.43X 10⁶IU/mg。

Drawings

FIG. 1 is a schematic diagram of the construction of pPIC 9K-CaIFN-. alpha.7 vector;

FIG. 2 shows the electrophoresis of linearized pPIC 9K-CaIFN-. alpha.7 plasmid, in lane 1, pPIC 9K-CaIFN-. alpha.7 plasmid, in lane 2, SacI-linearized pPIC 9K-CaIFN-. alpha.7 plasmid, M: DL5000DNA Marker;

FIG. 3 is a high copy transformant screening chart;

FIG. 4 shows the PCR identification result of recombinant strain GS115/pPIC 9K-CaIFN-alpha 7; in the figure, lanes 1-8 are PCR amplification products of 1-8 cloned genomic DNAs, 9 is PCR product of Pichia pastoris GS115/pPIC9K genomic DNA, M: DL2000DNA Marker;

FIG. 5 shows the SDS-PAGE identification of the expression product of recombinant GS115/pPIC 9K-CaIFN-alpha 7; lane 1 is the culture supernatant of Pichia pastoris GS115/pPIC9K induced for 72h, lanes 2-9 are the culture supernatant of clonal strain induced for 72h, and M is protein molecular weight Marker;

FIG. 6 shows the Western blot identification result of the expression product of recombinant strain GS115/pPIC 9K-CaIFN-alpha 7; lane 1-8 is the culture supernatant of the clone induced for 72h, Lane 9 is the culture supernatant of Pichia pastoris GS115/pPIC9K induced for 72h, M is the pre-stained protein molecular weight Marker;

FIG. 7 is an SDS-PAGE analysis of purified products of canine recombinant interferon-. alpha.7 protein; lane 1, flow-through, lane 3, 80mM imidazole eluate, M is protein molecular weight Marker;

FIG. 8 is a Western blot analysis chart of purified products of canine recombinant interferon- α 7 protein; 1 is culture supernatant obtained when pichia pastoris GS115/pPIC9K is induced for 72 hours, 2 is a purified sample, and M is a prestained protein molecular weight Marker;

FIG. 9 shows the results of detecting the biological activity of the expression product of recombinant GS115/pPIC 9K-CaIFN-alpha 7; c is cell control group, V is virus control group.

Detailed Description

The present invention will be described in detail with reference to examples.

Example 1 optimization of Gene sequence encoding Canine Interferon-alpha 7 and design and Synthesis of primers

The invention optimizes codons and synthesizes a target gene sequence according to the cDNA sequence (NM-001006654.1) of canine interferon-alpha 7 in GenBank as shown in SEQ ID No. 2. The codon of the gene encoding canine interferon-alpha 7 uses the codon most preferred by Pichia pastoris. The gene sequence of the optimized canine interferon-alpha 7 is shown as SEQ ID No.3, and the amino acid sequence of the coding protein is shown as SEQ ID No. 1.

Example 2 construction of pPIC 9K-CaIFN-. alpha.7 expression vector

The CaIFN-alpha 7 gene fragment shown in SEQ ID No.3 in the example 1 and the vector pPIC9K are subjected to double enzyme digestion by EcoRI and NotI, the double enzyme digestion fragment is recovered, and the CaIFN-alpha 7 gene fragment and the vector pPIC9K are subjected to double enzyme digestion according to the ratio of 3: 1 at 16 ℃ overnight, transformed into E.coli competent cells DH5a, plated on LB plates containing Amp (100ug/mL), and cultured at 37 ℃ for 16-24 h. Single colonies were picked, plasmids were extracted, PCR-identified and sent to the Producer corporation for sequencing. The pPIC 9K-CaIFN-. alpha.7 vector was constructed as shown in FIG. 1.

EXAMPLE 3 electroporation transformation of Yeast cells and selection of high expression transformants

Selecting a colony which is identified by PCR and sequenced correctly for amplification culture, selecting a high-purity plasmid large-extraction kit of Tiangen biochemical reagents, and extracting plasmids according to the instruction. Pichia pastoris GS115 competent cells were prepared according to the Invitrogen Pichia Expression Kit instructions. The recombinant plasmid was linearized with the restriction enzyme SacI and digested for 5h in a metal bath at 37 ℃. The linearization system is shown in Table 1.

TABLE 1

Reagent	Dosage (mu L)
		pPIC9K-CaIFN alpha 7 plasmid	About	1. mu.g
SacI	1μL
		Cut Smart buffer	2μL
Add dd H₂O to	50μL

The linearized plasmid was then recovered using ethanol precipitation. The linearized plasmid was added to 3mol/L NaAc (pH5.2) in 0.1 volume and absolute ethanol in 2.5 volumes, left at-20 ℃ for more than 30min, centrifuged at 14,000r/min for 20min at 4 ℃, and the supernatant was decanted. Adding 700 mu L of 75% ethanol for rinsing, centrifuging at high speed for a plurality of minutes, then removing the supernatant, and repeating the steps. The EP tube was inverted on a clean bench absorbent paper for about 10 minutes to remove as much water and residual ethanol as possible, and 20. mu.L of ddH2O was redissolved, quantified and placed on ice for further use. And the recovered plasmid concentration was determined by one-drop. The linearized and purified pPIC 9K-CaIFN-. alpha.7 plasmid was subjected to gel electrophoresis, and the results were in agreement with the expectations, and are shown in FIG. 2.

100 mu L of the prepared yeast allelochemicals GS115 are mixed with 10 mu L (10-15 mu g) of linearized recombinant expression plasmids, the mixture is transferred into a precooled electrotransformation cup with the depth of 0.2cm, the liquid is gently shaken to the bottom of the cup, and the cup is immediately iced for 5 min. Wiping water vapor, placing on an electric transfer instrument, setting electric transfer parameters, and selecting the electric transfer parameters: the voltage is 1500v, the capacitance is 25 muF, the resistance is 200 omega, the electric shock time is 4-5 ms, and the electric conversion is immediately carried out.

Immediately after electrotransfer, 1mL of precooled 1mol/L sorbitol is added into an electrotransfer cup, the mixture is quickly and uniformly mixed, the mixture is transferred into a new 1.5mL EP tube, and the mixture is incubated for 1-2 h in an incubator at 30 ℃ without shaking. And (3) coating 100 mu L of bacterial liquid on an MD (MD) plate, inversely culturing for 3-5 days in a 30 ℃ culture box, and observing the growth of transformants. The diameter of the bacterial colony is 1 mm. Meanwhile, a mixture of DNA and GS115 cells transformed without electric shock was plated as a negative control.

Single colonies growing on the plates were picked with a sterile toothpick, inoculated sequentially by photolithography onto YPD plates containing G148 at a concentration gradient of 0, 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0mg/mL, and high G418 resistant strains were selected step by step. The sequence of photocopies was from a YPD plate containing a higher concentration of G418 to a YPD plate containing a lower concentration of G418. The plate is placed in a constant temperature incubator at 30 ℃ for 3-5 days until a clone colony grows out (see figure 3).

Single colonies growing on high G418 YPD plates were picked up, and after picking with a pipette tip, the cells were dispersed in 10. mu.L of water, and the single colonies were stored by gently dipping the pipette tip onto a new pre-labeled MD plate. Boiling in boiling water bath for 10min, freezing in liquid nitrogen for 30min, boiling in boiling water bath for 10min, centrifuging at 12000r/min for 5min, taking supernatant as template, and performing PCR identification by respectively using a segment of sequence of target gene as upstream primer and downstream primer. The reaction system is shown in Table 2:

an upstream primer F: 5'-CGGAATTCATCACCATCACCATCACTGTC-3', respectively;

a downstream primer R: 5'-TTGCGGCCGCTTACTCCTTTCTTCTAATTC-3', respectively;

TABLE 2

PCR reaction system	Volume (μ L)
		2X Premix	10
Forward Primer 10μM	1
		Reverse Primer 10μM	1
Form panel	0.5
		Add dd H₂O to	20

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; 30 cycles of 94 ℃ for 1min, 60 ℃ for 30s and 72 ℃ for 30 s; finally, extension is carried out for 10min at 72 ℃.

And (3) carrying out electrophoresis on the PCR product by using 1.0% agarose gel nucleic acid, and observing an electrophoresis result under an ultraviolet gel imager. As shown in fig. 4. The results showed that the PCR products of the 8 clones selected were consistent with the expected size.

Example 4 inducible expression and identification of recombinant Yeast engineered bacteria

The recombinant strain GS115/pPIC 9K-CaIFN-alpha 7 and the empty vector transformed yeast GS115/pPIC9K monoclonal antibodies of 8 positive clones obtained in the embodiment 3 are respectively inoculated in 20-30mL BMGY culture medium, cultured at 30 ℃ and 220rpm/min for about 16-20 h until the OD600 value of the thallus is 2-6 and 1500-3000 g, centrifuged at room temperature for 10min, the culture medium is discarded, and the thallus is collected. And (3) resuspending the thallus precipitate by using 25mL of BMMY culture medium, culturing for 4-5 days at 28 ℃ and 220rpm/min with the OD600 value of 1-2, and supplementing methanol every 24 hours until the final concentration is 1%.

After induction culture is finished, collecting culture supernatant, adding a sample into a protein Loading buffer, boiling for 10min at 100 ℃, taking 10 mu L of sample, and carrying out SDS-PAGE electrophoresis and western blot detection. The results of SDS-PAGE (FIG. 5) and Western blot (FIG. 6) showed that: the secretion supernatant can see obvious target bands at about 24KD and 27KD, because pichia pastoris has the effect of post-translational modification on exogenous recombinant protein, glycosylation modification can be carried out on the recombinant protein, the problem of incomplete N-glycosylation modification can exist, and the recombinant canine interferon has two N-glycosylation sites (at positions 78-80 and 133-135), so that two bands with different relative molecular weights are obtained due to incomplete glycosylation modification. Based on the expression level of clones 1-8, clone No. 5 was selected for subsequent experiments.

Example 5 purification of recombinant pPIC9K-CaIFN-a7 and protein quantification by Coomassie Brilliant blue method

Clone No. 5 was inoculated into 5mL YPD seed solution, cultured overnight, and then transferred to 50mL BMGY, which was cultured for 16-20 h at 30 ℃ with shaking table 220 r/min. Centrifuging 1500-3000 g of a sterilized centrifuge tube at normal temperature for 10min, diluting the thallus with an induction culture medium BMMY to an OD600 value of 1-2, culturing at 28 ℃ and 220rpm/min for 4-5 d, and supplementing methanol every 24h until the final concentration is 1%. After the induction culture is finished, collecting culture supernatant, filtering the culture supernatant by a 0.45 mu m filter, and performing affinity chromatography by using an affinity chromatography column, wherein the method comprises the following specific steps:

washing: cleaning of Ni with pure Water²⁺Chelating affinity chromatographic column and instrument pipeline.

Balancing: after 10 column volumes of pure water, 3-5 column volumes of Buffer A (50mM Tris,100mM NaCl pH7.4) were equilibrated.

Loading: and when the conductivity value and the absorption value of 280nm wavelength are detected stably on line, the sample injection is started.

Balancing: after loading, the column was chromatographed using Buffer A, and unbound heteroproteins were washed off.

And (3) elution: the target protein was collected by washing with an Elution Buffer (50mM Tris,100mM NaCl,500mM Imidazole, pH7.4) containing Imidazole at various concentrations.

Fractions collected during purification were analyzed by SDS-PAGE, see FIG. 7. SDS-PAGE detection shows that: the molecular weight of the canine recombinant interferon-alpha 7 protein expressed by the recombinant strain is about 24KD, and the molecular weight is consistent with the size of the expected target protein. The results of scanning analysis by using gel imaging system software show that the expression amount of the target protein accounts for 50% of the total protein of the thallus, the expression amount is better, and the protein yield is 138 mg.

Dialyzing the eluted components into a pH7.4Tris-HCl buffer system, detecting protein quantification by a Coomassie brilliant blue method, and controlling the concentration of the purified canine recombinant interferon-alpha 7 protein to be 0.72 mg/mL.

Example 6 Western blot identification of expression products

Adding the purified canine recombinant interferon-alpha 7 into a protein loading buffer, boiling for 10min at 100 ℃, taking 10 mu L of boiled sample, loading the sample, selecting 15% SDS-PAGE gel for electrophoresis, and electrically transferring the polyacrylamide gel subjected to electrophoresis to a PVDF membrane by an electrowetting transfer system at the parameter of 300mA for 60 min. Taking out the PVDF membrane, sealing the PVDF membrane in 5% skimmed milk powder TBST at 37 ℃ for 1h, and discarding the liquid; washing the membrane with TBST, adding Anti-His monoclonal antibody, and incubating overnight at 4 ℃; washing the membrane with TBST for 3 times, 8min each time, adding HRP-goat anti-mouse monoclonal antibody diluted with 1:10000, and incubating at 37 deg.C for 1 h; washing the membrane with TBST for 3 times, 8min each time, directly developing with DAB developing solution, and taking pictures and recording. As shown in fig. 8. The Western-blot results show that: the expression product can be specifically combined with the monoclonal antibody, has obvious reaction bands at 24KD and 27KD, and is consistent with the result of SDS-PAGE, which shows that the expression product has good immunoreactivity.

Example 7 detection of Canine recombinant Interferon-alpha 7 bioactivity

Antiviral Activity of the purified Canine recombinant Interferon- α 7 of example 5 was determined on MDCK-VSV System using the Microcytopathy inhibition method

Preparing a solution of a to-be-detected product: and sequentially carrying out 2-fold gradient dilution on the interferon in a 96-well cell culture plate from 1: 20000, wherein each dilution is carried out with one multiple hole, and 10 dilutions (1-10 holes) are carried out in total.

Removing culture medium in the full monolayer cell bottle, washing with PBS for 2 times, digesting, collecting cells, and preparing into 3.0 × 10/ml with nutrient solution⁵～4.0×10⁵Cell suspensions of individual cells were inoculated into the above 96-well cell culture plate, 100. mu.l per well, and cultured at 37 ℃ under 5% CO2 for 24 hours; discarding supernatant in cell culture plate, diluting VSV virus solution for use (-80 deg.C storage) with maintenance solution to 100TCID50/0.1mL, adding 100 μ l per well into each well of cell plate, placing into 37 deg.C, 5% CO2 incubator, setting No. 11 well as cell control C, adding neither interferon protection nor VSV virus solution, and adding cell suspension only; well 12 is virus control V, no interferon protection, and VSV virus fluid only.

Culturing for 18-24 h, and observing half cell lesion number to show the most 50% of cell lesionsThe high dilution is one Interferon Unit (IU). The activity of canine interferon alpha 7 recombinant protein (IU/mg) was calculated according to the Reed-muench method. The result shows that the antiviral activity of the canine interferon alpha 7 recombinant protein is 1.03 multiplied by 10⁶IU/mL (see FIG. 9), specific activity 1.43X 10⁶IU/mg. In conclusion, the canine interferon alpha 7 recombinant protein has better in vitro antiviral activity.

The above detailed description of the canine recombinant interferon alpha 7, the preparation method and use thereof, the expression vector containing the canine recombinant interferon alpha 7 and the host cell with reference to the examples is illustrative and not restrictive, and several examples can be cited within the scope of the present invention, so that variations and modifications thereof without departing from the general concept of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

<110> Jiuzhuan Biotechnology Co., Ltd, Anhui; tu lake InteFell BioProcessary research institute Co Ltd

<120> canine recombinant interferon alpha 7, preparation method and application thereof, expression vector containing canine recombinant interferon alpha 7 and application thereof

Host cell

<130> 1

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 510

<212> PRT

<213> amino acid sequence of canine recombinant interferon alpha 7

<400> 1

1 His His His His His His Cys His Leu Pro Asp Thr His Gly Leu Arg Asn Trp Arg Val

21 Leu Thr Leu Leu Gly Gln Met Arg Arg Leu Ser Ala Gly Ser Cys Asp His Tyr Thr Asn

41 Asp Phe Ala Phe Pro Lys Glu Leu Phe Asp Gly Gln Arg Leu Gln Glu Ala Gln Ala Leu

61 Ser Val Val His Val Met Thr Gln Lys Val Phe His Leu Phe Cys Pro Asp Thr Ser Ser

81 Ala Pro Trp Asn Met Thr Leu Leu Glu Glu Leu Cys Ser Gly Leu Ser Glu Gln Leu Asp

101 Asp Leu Glu Ala Cys Pro Leu Gln Glu Ala Gly Leu Ala Glu Thr Pro Leu Met His Glu

121 Asp Ser Thr Leu Arg Thr Tyr Phe Gln Arg Ile Ser Leu Tyr Leu Gln Asp Arg Asn His

141 Ser Pro Cys Ala Trp Glu Met Val Arg Ala Glu Ile Gly Arg Ser Phe Phe Ser Ser Thr

161 Ile Leu Gln Glu Arg Ile Arg Arg Lys Glu

<210> 2

<211> 492

<212> DNA

<213> Gene sequence before optimization of canine recombinant interferon alpha 7

<400> 2

cacctgcccg acacccacgg cctgcgcaac tggagggtcc tgacgctcct gggacagatg 60

aggagactct ccgccggctc ttgtgaccac tacaccaatg actttgcctt ccccaaggag 120

ctgtttgatg gccagcggct ccaggaggcg caggccctct ctgtggtcca cgtgatgacc 180

cagaaggtct tccacctctt ctgcccggac acgtcctctg ctccttggaa catgactctc 240

ctggaggaac tgtgctcggg gctctctgag cagctggatg acctggaggc ctgtcccctg 300

caggaggcgg ggctggccga gacccccctc atgcatgagg actccaccct gaggacctac 360

ttccaaagga tctccctcta cctgcaagac aggaaccaca gcccgtgtgc ctgggagatg 420

gtccgagcag aaatcgggag atccttcttc tcctcgacaa tcttgcaaga aagaatcagg 480

aggaaggaat ga 492

<210> 3

<211> 510

<212> DNA

<213> optimized gene sequence of canine recombinant interferon alpha 7

<400> 3

catcaccatc accatcactg tcatttgcca gatactcacg gtttgagaaa ctggagagtt 60

ttgactttgt tgggtcaaat gagaagattg tctgctggtt cttgtgatca ttacactaac 120

gatttcgctt tccctaagga attgtttgat ggtcaaagat tgcaagaggc tcaagctttg 180

tctgttgttc atgttatgac tcaaaaggtt ttccacttgt tctgtccaga tacttcttct 240

gctccttgga acatgacttt gttggaagag ttgtgttctg gtttgtctga acaattggat 300

gatttggagg cttgtccatt gcaagaagct ggtttggctg agactccttt gatgcatgaa 360

gattctactt tgagaactta cttccaaaga atctctttgt atttgcaaga tagaaatcac 420

tctccatgtg cttgggaaat ggttagagct gagatcggta gatctttctt ttcttctact 480

atcttgcaag aaagaattag aagaaaggag 510

Claims

1. A canine interferon alpha 7 gene is characterized in that the nucleotide sequence of the canine interferon alpha 7 gene is shown as SEQ ID No. 3.

2. A recombinant expression vector comprising the canine interferon alpha 7 gene according to claim 1.

3. A host cell comprising the canine interferon alpha 7 gene of claim 1 or the recombinant expression vector of claim 2.

4. The host cell of claim 3, wherein the host cell is Pichia pastoris cell strain GS 115.

5. A preparation method of canine interferon alpha 7 protein is characterized by comprising the following steps:

(1) connecting the canine interferon alpha 7 gene of claim 1 with a vector pPIC9K, transforming into an escherichia coli competent cell DH5a, culturing, picking out a single colony, extracting a plasmid, and performing PCR identification;

(3) electrically transforming the recombinant plasmid obtained in the step (2) into a pichia pastoris cell strain GS115, and sequentially carrying out incubation and culture until the diameter of a bacterial colony is 1 mm;

(4) culturing the recombinant strain GS115/pPIC9K-CaIFN alpha 7 of the positive clone bacteria obtained in the step (3) in a BMGY culture medium until the bacteria OD₆₀₀Centrifuging to obtain cell pellet with value of 2-6, and diluting the cell pellet with BMMY culture medium to OD₆₀₀The value is 1-2, culturing for 4-5 days, supplementing methanol every 24 hours until the final concentration is 1%, performing induction culture, and centrifuging to collect supernatant; after purification, the canine interferon alpha 7 protein can be obtained.

6. The use of the canine interferon alpha 7 protein prepared by the preparation method according to claim 5 in the preparation of anti-VSV virus drugs.

7. A biological product containing canine interferon alpha 7 protein prepared by the preparation method according to claim 5.