CN103450355B - recombinant bovine alpha interferon and application thereof - Google Patents

Abstract

The present invention relates to recombinant bovine alpha interferon, the cattle IFN-α G subtype gene (Y-BoIFN-α G) that its corresponding gene is the Niu Gongyou IFN-α gene (Y-CoBoIFN-α) that optimizes of Niu Gongyou IFN-α gene (CoBoIFN-α), genetic codon and genetic codon optimizes, and the albumen encoded is at the biological activity of anti-virus aspect.The present invention has designed and synthesized CoBoIFN-α gene first, and the first identified antiviral activity of CoBoIFN-α and BoIFN-α G, result confirms that the antiviral activity of BoIFN-α-G and CoBoIFN-α is at least above other similar ten times, is more suitable for clinical practice and industrialized developing.

Description

Recombinant bovine alpha interferon and application thereof

Technical field

The present invention relates to recombinant bovine alpha interferon and application thereof, be specifically related to Niu Gongyou IFN-α (CoBoIFN-α), the nucleotide sequence of cattle IFN-α G hypotype (Y-BoIFN-α G) of Niu Gongyou IFN-α (Y-CoBoIFN-α) that genetic codon optimizes and genetic codon optimization and expressing protein thereof, and its biological activity, belong to biology field.

Background technology

Cattle IFN-α is by the protein families of more than 10 correlation function gene codes, by one open reading frame (ORF) of 567 nucleotide codings, ripe cattle IFN-α is made up of 166 aminoacid, induces the signal peptide of its secreting, expressing to be made up of 23 aminoacid.The same with humanIFN-α, cattle IFN-α has 4 conservative cysteine, lays respectively at the 1st, 29,98 and 138, two intramolecular disulfide bond (Cys1-Cys98 can be formed, Cys29-Cys138), cattle IFN-α does not have glycosylation site in its natural state.

As far back as 1985, cattle IFN-α gene and cattle IFN-α were just once done detailed research by Velan etc..Velan etc. have gone out cattle IFN-α gene with humanIFN-α's gene for probe separates from the cDNA library of cattle, speculate that this gene was including at least 10-12 hypotype at that time, to A therein, B, tetra-hypotypes of C, D have checked order, and cattle IFN-α C hypotype have been expressed in E. coli.1996, Chaplin etc. separated cattle IFN-α E hypotype from the calf enterocyte of rotavirus infection, and has expressed in insect cell with baculovirus AcMNPV for carrier, and the recombiant protein of expression has obvious antiviral activity on bovine kidney cells.Paul etc. have cloned again cattle IFN-α G, F, H hypotype from the Intestinum Bovis seu Bubali epithelial cell of rotavirus infection subsequently, and have checked order.

Cattle IFN-α contains 10-12 hypotype, and between different subtype, homology is significantly high, and eight the hypotype nucleotide of cattle IFN-α A, B, C, D, E, F, G, H reported and amino acid identity have all exceeded more than 90%.Different subtype is probably by different cells or what same cell secrete at different times, but due to the comparative study of shortage system, whether its biologic activity is variant not clear at present.

Total IFN-α, has another name called mutual IFN-α, is non-natural I type interferon of a kind of restructuring, and it is to be set up gene by the synthesis of the original mutual Amino acid profile of each position of IFN-α family.YitzhakStabinsky et al. designs according to the consensus sequence of humanIFN-α's gene family and has cloned a kind of artificial IFN-α being called total IFN-α.Research finds the homology respectively 89%, 30% and 60% of total IFN-α and IFN-α, IFN-β, IFN-ω.LiHuang et al. is by scanning the sequence of 17 kinds of natural pig IFN-α hypotypes, and the combination of amino acids the most often occurred each relevant position, successful design has also synthesized the total IFN-α of a boar.Research finds that the total antiviral of IFN-α, cell proliferation, promotion NK activity, inducing cytokine produce and the natural IFN-α of energy force rate of inducible gene expression is high.Cattle genome sequencing plan is complete, therefore engineer synthesize Niu Gongyou IFN-α and have important production and using value.

Have been found that cattle IFN-α can resist propagation and the duplication of the multiple viruses such as African swine fever virus, foot and mouth disease virus, viral diarrhea virus, vesicular stomatitis virus, infectious bovine rhinotrachetis virus, bovine rota up to now；And the research on people with pig shows, compared with total IFN-α is similar with it, CoIFN-α because of antiviral be obviously enhanced, side effect is less and is more suitable for clinical practice, therefore Niu Gongyou IFN-α gene is designed and synthesized, and suddenlyd change become with it only difference one amino acid whose cattle IFN-α G subtype gene (two kinds of genes are the novel cattle IFN-α being never investigated), analyze its antiviral activity, significant exploration can be provided for development of new cattle antiviral interferon preparation.

Summary of the invention

The present invention relates to recombinant bovine alpha interferon Niu Gongyou IFN-α gene, optimize its genetic codon, to improve expressing quantity, the sudden change of Niu Gongyou IFN-α is become with it only one amino acid whose cattle IFN-α G subtype gene of difference, utilize pichia yeast expression system to express artificial gene without any modification sequence (Niu Gongyou IFN-α, the Niu Gongyou IFN-α of gene optimization and cattle IFN-α G hypotype), and measure its antiviral activity.

The present invention relates to recombinant bovine alpha interferon CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G, aminoacid sequence respectively SEQIDNo.32, SEQIDNo.34 and the SEQIDNo.36 of described interferon CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G.

Encode nucleotide sequence respectively SEQIDNo.31, SEQIDNo.33 and the SEQIDNo.35 of above-mentioned interferon CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G.

Present invention additionally comprises the expression vector containing above-mentioned nucleotide sequence and the host cell containing described expression vector.

Additionally, present invention additionally comprises above-mentioned aminoacid sequence and nucleotides sequence is listed in the application in the medicine of preparation treatment or prevention cattle disease viral disease.The virus causing described cattle disease viral disease includes the multiple RNA viruses such as vesicular stomatitis virus, viral diarrhea virus.

CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G achieve highly-soluble secreting, expressing in Pichia sp., expression is 0.382mg/mL, 0.579mg/mL, 0.564mg/mL respectively, and records the antiviral activity of CoBoIFN-α, Y-CoBoIFN-α, Y-BoIFN-α G and cattle IFN-α A hypotype (BoIFN-α A) respectively 8.98 × 10 by cytopathic-effect inhibition assay⁷U/mg、9.16×10⁷U/mg、9.12×10⁷U/mg、8.93×10⁶U/mg.As a result, it was confirmed that by gene optimization, the Y-CoBoIFN-α expression than CoBoIFN-α improves 0.5 times, statistical analysis display CoBoIFN-α and BoIFN-α G antiviral activity are without significant difference, and relatively BoIFN-α A is high 10 times.The invention demonstrates that the antiviral activity of BoIFN-α-G and CoBoIFN-α is significantly higher than that other are similar, is more suitable for clinical practice and industrialized developing, and makes BoIFN-α-G and CoBoIFN-α obtain high efficient expression by genetic codon optimization.

Accompanying drawing explanation

Fig. 1 is the structure schematic diagram of CoBoIFN-α；

Fig. 2 is the structure schematic diagram of Y-BoIFN-α G；

Fig. 3 is the pcr amplification result figure of fragment A, B, C, D；

Fig. 4 is CoBoIFN-α gene PCR amplification；

Fig. 5 is the percent optimum results figure of Codon usage: wherein, and A is before CoBoIFN-α optimizes, B

After optimizing for CoBoIFN-α, C is before BoIFN-α G optimizes, and D is after BoIFN-α G optimizes；

Fig. 6 is the total IFN-α of cattle after optimizing and cattle IFN-α G subtype gene pcr amplification result figure；Wherein M is DNA molecular quality standard；1 is Y-CoBoIFN-α gene PCR amplification；2 is Y-BoIFN-α G gene PCR amplification；

Fig. 7 is plasmid pPICZ α A-CoBoIFN-α, pPICZ α A-Y-CoBoIFN-α and pPICZ α A-Y-BoIFN-α G qualification result figure；Wherein, M is DNA molecular quality standard；1,3,5 is XhoI single endonuclease digestion qualification result；2,4,6 is XhoI and XbaI double digestion qualification result；

Fig. 8 is the PCR qualification result figure of recombinant yeast pichia pastoris；Wherein, M is DNA molecular quality standard；1,11 is negative control；2,12 is positive control；3-10 is the CoBoIFN-α gene PCR amplification of eight strain difference Pichia sp.；13 is Y-CoBoIFN-α gene PCR amplification；14 is Y-BoIFN-α G gene PCR amplification；

Fig. 9 is the comparison diagram of eight strain expression of recombinant yeast amounts；Wherein, after 6,10: empty carrier GS115-pPICZ α A induction；1:1 bacterial strain；2:2 bacterial strain；3:3 bacterial strain；4:4 bacterial strain；5:5 bacterial strain；7:6 bacterial strain；8:7 bacterial strain；9:8 bacterial strain；

Figure 10 is eight strain expression of recombinant yeast albumen indirect ELISA detection figure；

Figure 11 is the SDS-PAGE expression spirogram analyzing each time point of yeast secreted protein；Wherein, M. protein standard；After 1: empty carrier GS115-pPICZ α A induction；Secretory protein after 2:12h；Secretory protein after 3:24h；Secretory protein after 4:36h；Secretory protein after 5:48h；Secretory protein after 6:60h；Secretory protein after 7:72h；Secretory protein after 8:84h；Secretory protein after 9:96h；

The indirect ELISA that Figure 12 is each time point expression of recombinant yeast albumen analyzes result figure；

Figure 13 is the SDS-PAGE expression of results figure analyzing yeast secreted protein；Wherein, M is protein standard；2,4,5 is after empty carrier pPICZ α A induction；1 is secretory protein after induction G-p-CoBoIFN-α 96h；3 is secretory protein after induction G-p-Y-CoBoIFN-α 96h；6 is secretory protein after induction G-p-Y-BoIFN-α G96h；

Figure 14 is expression of recombinant yeast albumen indirect ELISA detection figure；

Figure 15 is the Westernblot analysis chart of yeast secreted protein；Wherein, M is protein standard;1,3,5 is empty carrier negative control；2 is the secretory protein CoBoIFN-α of yeast expression；4 is the secretory protein Y-CoBoIFN-α of yeast expression；6 is the secretory protein Y-BoIFN-α G of yeast expression；

Figure 16 is that yeast secreted protein is to the VSV inhibitory action figure bred；

Figure 17 is that yeast secreted protein Y-CoBoIFN-α is to the VSV inhibitory action figure bred；

Figure 18 is that yeast secreted protein Y-BoIFN-α G is to the VSV inhibitory action figure bred；

Figure 19 is that the anti-BoIFN-alpha immunization polyvalent antibody of rabbit blocks CoBoIFN-α, Y-CoBoIFN-α, Y-BoIFN-α G antiviral activity curve chart.

Detailed description of the invention

Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and apparent.But embodiment is only exemplary, the scope of the present invention is not constituted any restriction.It will be understood by those skilled in the art that and the details of technical solution of the present invention and form can be modified or replace lower without departing from the spirit and scope of the present invention, but these amendments and replacement each fall within protection scope of the present invention.

Test material:

1 plasmid, Host Strains, virus

Host Strains E.coilTG1, DH5 α preserves by microbiological inhibitory teaching and research room of animal medicine institute of Northeast Agricultural University.Vesicular stomatitis virus (VSV) is so kind as to give by animal medicine institute of Northeast Agricultural University teacher Ren Xiaofeng.Pichia pastoris GS115 is so kind as to give by the Chinese Academy of Agricultural Sciences Harbin veterinary institute Gao Honglei researcher.

2 cells, serum, albumen

Bovine kidney cells (MDBK) cell strain, rabbit anti-BoIFN-α polyclonal antibody, yeast secreted expression protein B oIFN-α A, the BoIFN-each subtype gene of α multigene family: pMD18-T-BoIFN-α-1, pMD18-T-BoIFN-α-6, pMD18-T-BoIFN-α-7 after purification, pMD18-T-BoIFN-α-14 is by this laboratory clone and preserves.

The acquisition of embodiment 1 PROTEIN C oBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G

The gene optimization of the design of 1.CoBoIFN-α and CoBoIFN-α and BoIFN-α G and synthesis

The design of 1.1CoBoIFN-α gene order

CoBoIFN-α is the IFN-α heterozygote of the non-natural appearance of a kind of synthesis, its aminoacid sequence is by scanning all IFN-α subtype sequences announced in GenBank, being designed by the combination of amino acids that each relevant position the most often occurs, its all aminoacid are both from each subtype gene sequence of BoIFN-α family.In this example, the DNA sequence of CoBoIFN-α is the method adopting fusion DNA vaccine, the each respective segments (A, B, C, D) utilizing clone gene BoIFN-α-1, BoIFN-α-6, BoIFN-α-7 and BoIFN-α-14 is spliced, but the acquisition of the DNA of CoBoIFN-α is not limited to that example.

The design of 1.2 primers and synthesis

Owing to adopting the method synthesis CoBoIFN-α of fusion DNA vaccine, so all there is the spacer end of 18-25bp between adjacent primer；In order to make recombiant protein be secreted into extracellular, therefore supplement the base sequence AAAAGA of protease (Kex2) restriction enzyme site on pPICZ α A carrier when design connects the forward primer of Yeast expression carrier, and XhoI and XbaI restriction enzyme enzyme recognition site is introduced respectively in primer upstream, downstream, in order to gene is connected on expression vector pPICZ α A.Each primer is as follows:

(1) the fusion DNA vaccine primer synthesizing CoBoIFN-α is SEQIDNo.3-SEQIDNo.8, and wherein, SEQIDNo.3 is Aa2, SEQIDNo.4 be Bs1, SEQIDNo.5 be Bs2, SEQIDNo.6 be Cs1, SEQIDNo.7 be Ca2, SEQIDNo.8 is Ds1.

(2) primer connecting Yeast expression carrier is SEQIDNo.9 and SEQIDNo.10, wherein: SEQIDNo.9 is Ye-BoIF5S, SEQIDNo.10 is Ye-BoIF3a.

The pcr amplification of 1.3CoBoIFN-α

The fusion DNA vaccine amplification of CoBoIFN-α includes three reactions steps:

(1) independent amplification of fragment to be fused；

(2) each intersegmental fusion reaction, adds the purified product of first step reaction, and without primer, by 10 circulations, renaturation extends the lap between fragment；

(3) total length of each fusion fragment is expanded, the non-purified product that second step is reacted, add Outside primer, carry out 25 circulations.

Fig. 1 is shown in by the pcr amplification schematic diagram of CoBoIFN-α.

The gene optimization of 1.4CoBoIFN-α and BoIFN-α G

By software under the constant premise of the aminoacid sequence of Niu Gongyou CoBoIFN-α and BoIFN-α G, by changing the methods such as the codon adaptation indexI of sequence, G/C content, mRNA secondary structure and Codon usage, carry out CoBoIFN-α and the gene optimization of BoIFN-α G, to improving the expression of CoBoIFN-α and BoIFN-α G.

The design of 1.5 primers and synthesis

Sequence after optimizing is split into 16 small fragments, as primer after synthesis, between adjacent primer, all there is the spacer end of 20-25bp.Owing to the gene order of BoIFN-α G and CoBoIFN-α only differs an aminoacid, so the BoIFN-α G gene order after only needing additionally two primers of many designs to be optimized.Each primer is SEQIDNo.11-28, and wherein SEQIDNo.11 is p1, SEQIDNo.12 be p2, SEQIDNo.13 be p3, SEQIDNo.14 be p4, SEQIDNo.15 be p5, SEQIDNo.16 be p6, SEQIDNo.17 be p7, SEQIDNo.18 be p8, SEQIDNo.19 be p9, SEQIDNo.20 be p10, SEQIDNo.21 be p11, SEQIDNo.22 be p12, SEQIDNo.23 be p13, SEQIDNo.24 be p14, SEQIDNo.25 be p15, SEQIDNo.26 be p16, SEQIDNo.27 be GL7, SEQIDNo.28 is GL8.The synthesis of 1.6CoBoIFN-α and BoIFN-α G optimized gene

The method that the synthesis of Y-CoBoIFN-α adopts Overlap extension PCR, is first divided into four groups by each adjacent primer, by 10 circulations, extends the lap between fragment；By first step product, without primer, by 10 circulations, continue to extend the lap between fragment；Then the product reacted by second step, adds two ends primer (p1, p16), carries out 25 circulations, obtain Y-CoBoIFN-α, replaces p7 and p8 with GL7 and GL8, obtains Y-BoIFN-α G by same Overlap extension PCR method.

Y-BoIFN-α GPCR expands schematic diagram and sees Fig. 2.

The structure of 2 recombination yeasts containing CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G and expression

The structure of 2.1 yeast secreted expression carrier pPICZ α A-CoBoIFN-α, pPICZ α A-Y-CoBoIFN-α and pPICZ α A-Y-BoIFN-α G

(1) preparation of CoBoIFN-α, Y-CoBoIFN-α, Y-BoIFN-α G gene and carrier pPICZ α A double digestion purified product

Carrying out double digestion with XhoI and XbaI restricted enzyme respectively by reclaiming the PCR primer CoBoIFN-α, Y-CoBoIFN-α, Y-BoIFN-α G and the pPICZ α A expression vector that obtain, enzyme action system is as follows:

The total system of endonuclease reaction is 50 μ L, after 37 DEG C of water-bath enzyme action 2.5h, takes 2 μ L digestion products and carries out electrophoresis on 0.8% agarose gel, observes enzyme action effect, if respond well, glue reclaims purpose fragment, be stored in-20 DEG C standby.

(2) CoBoIFN-α, Y-CoBoIFN-α, Y-BoIFN-α G gene link with carrier pPICZ α A's

Double digestion PCR primer after purification being attached with expression vector pPICZ α A respectively, linked system is as follows:

Overall reaction system is 10 μ L, and gently after mixing, metal bath 16 DEG C overnight, connects product and is respectively designated as pPICZ α A-CoBoIFN-α, pPICZ α A-Y-CoBoIFN-α and pPICZ α A-Y-BoIFN-α G.

(3) conversion of product is connected

From-70 DEG C of refrigerators, take out three centrifuge tubes equipped with DH5 α competent cell, be put on ice for treating that it melts；Connect product by three kinds to mix gently with competent cell respectively, ice bath 30min；Put into heat shock 1min30s in the water bath with thermostatic control of 42 DEG C；It is transferred quickly on ice, ice bath 3min-5min；Being separately added into 200 μ L LB liquid medium in every centrifuge tube, in 37 DEG C of shaking tables, concussion is cultivated after 1h and is spread evenly across respectively containing zeocin(25 μ g/mL) LB flat board on；Put in 37 DEG C of constant incubators, be inverted after liquid is absorbed and cultivate 14h-18h.

(4) extraction of recombiant plasmid

Picking 4-6 medium sized bacterium colony of difference on three pieces of solid LB flat boards, it is inoculated in 5mL respectively and contains in the antibiotic LB liquid medium of zeocin, it is placed in overnight incubation in 37 DEG C of shaking tables, collect thalline, extract recombiant plasmid according to the mini prep method of plasmid DNA " the SDS alkaline lysis prepare " in the third edition " Molecular Cloning: A Laboratory guide ".

(5) qualification of recombiant plasmid

The recombiant plasmid XhoI of extraction is carried out single endonuclease digestion qualification, carries out double digestion qualification with XbaI and XhoI.

XhoI single endonuclease digestion identification system:

XbaI and XhoI double digestion reaction system is as follows:

Reacting total system and be 10 μ L, mixing is placed on enzyme action 2h in 37 DEG C of waters bath with thermostatic control gently, takes 5 μ L digestion products and on 0.8% agarose gel, enzyme action result is observed.

Recombiant plasmid respectively through single, double cut qualification after, it is thus achieved that positive plasmid called after pPICZ α A-CoBoIFN-α, pPICZ α A-Y-CoBoIFN-α and pPICZ α A-Y-BoIFN-α G.Positive recombiant plasmid is stored in-20 DEG C, and takes fresh positive bacterium solution and deliver Shenzhen Huada Genetic Technology Co., Ltd and carry out sequencing.

2.2 build the recombinant yeast pichia pastoris containing CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G gene

(1) linearisation of Yeast expression carrier

Take positive plasmid pPICZ α A-CoBoIFN-α, pPICZ α A-Y-CoBoIFN-α, pPICZ α A-Y-BoIFN-α G, carry out single endonuclease digestion with SacI restricted enzyme respectively.

SacI single endonuclease digestion reaction system is as follows:

Overall reaction system is 100 μ L, 37 DEG C of water bath with thermostatic control enzyme action 2.5h, takes 2 μ L digestion products and detects whether that enzyme action is complete on 0.8% agarose gel.Precipitate with ethanol reclaims enzyme action linearized fragment completely, is dissolved in deionized water, measures the concentration of linearized fragment, and is diluted to 5ug/20 μ L-15ug/20 μ L with deionized water.

(2) preparation of Pichia sp. electrocompetent

With Pichia pastoris GS115 strain at the flat lining out of non-resistant YPD, it is placed in 30 DEG C of incubators and cultivates 48h；Single colony inoculation on picking flat board is in 5mLYPD fluid medium, and 29 DEG C of 220r/min constant-temperature tables cultivate 24h；Taking 200 μ L to transfer in containing in 100mLYPD triangular flask, 29 DEG C of 220r/min constant-temperature tables cultivate 14h, are about 1.3 to OD600nm；4 DEG C of centrifugal 5min of 3800r/min, abandon supernatant；With the sterilized water of 100mL pre-cooling, thalline is resuspended.4 DEG C of centrifugal 5min of 3800r/min, abandon supernatant.With the sterilized water of 50mL pre-cooling, thalline is resuspended；4 DEG C of centrifugal 5min of 3800r/min, abandon supernatant；With the sorbitol (1mol/L) of 10mL pre-cooling, thalline is resuspended；4 DEG C of centrifugal 5min of 3800r/min, abandon supernatant；With the sorbitol (1mol/L) of 600ul pre-cooling, thalline is resuspended, in order to converting.

(3) Pichia sp. is electroporated

Take 80ul competent cell, add 20ul linearization plasmid DNA(15ug), gently after mixing, transfer to 0.2cm and shock by electricity in cup, after being placed in pre-cooling 5min, 1500v voltage click on ice, add 1mL sorbitol immediately.Electric shock cup inclusions is moved in 1.5mL sterile centrifugation tube, put into 30 DEG C of incubator quiescent culture 1h, after adding 1mLYPD, put into 30 DEG C of shaking table concussions and cultivate 1h, take 200uL and be uniformly applied on YPD flat board (concentration of Zeocin is 200 μ g/mL), cultivate 48h for 30 DEG C.The positive transformant of growth on YPD flat board is respectively designated as G-P-CoBoIFN-α, G-P-Y-CoBoIFN-α, G-P-Y-BoIFN-α G.(4) PCR of recombination yeast G-p-CoBoIFN-α, G-p-Y-CoBoIFN-α and G-p-Y-BoIFN-α G identifies

There is a pair universal primer at the two ends that genes of interest inserts on Yeast expression carrier pPICZ α A, this universal primer and specific primer Ye-BoIF5S, Ye-BoIF3aPCR is utilized to identify recombinant yeast pichia pastoris, universal primer sequence is SEQIDNo.29 and SEQIDNo.30, and wherein SEQIDNo.29 is 5 ' AOX1, SEQIDNo.29 is 3 ' AOX1.

Picking list bacterium colony is 200 μ g/mL in 5mLYPD(zeocin content) in, after 30 DEG C of constant-temperature tables cultivate 24h, adopt boil-freeze-cooking method extracts Yeast genome as the PCR template identified.Concrete grammar is as follows: take the centrifugal 5min of 1mL bacterium solution 8000rpm, stay thalline, thalline is washed 2 times with PBS, then with the 100 resuspended thalline of μ lTE, put into 10min in boiling water, be immediately placed in-70 DEG C of 30min, then place into 10min in boiling water, 8000rpm is centrifuged 10min, draws supernatant and is recombination yeast genome.

The PCR identification reaction system of eight strain recombinant yeast pichia pastoris G-p-CoBoIFN-α is as follows:

Recombination yeast genome 1.0 μ L；RTaqDNA polymerase 0.2 μ L；10 × PCRBuffer2.0 μ L；5 ' AOX1(10 μm of ol/L) 0.5 μ L；Ye-BoIF3a(10 μm of ol/L) 0.5 μ L；DNTP2.0 μ L；DdH2O13.8 μ L.

The PCR identification reaction system of recombination yeast G-p-Y-CoBoIFN-α and G-p-BoIFN-α G is as follows:

Recombination yeast genome 1.0 μ L；10 × PCRBuffer2.0 μ L；DNTP2.0 μ L；Ye-BoIF5S(10 μm of ol/L) 0.5 μ L；Ye-BoIF3a(10 μm of ol/L) 0.5 μ L；RTaqDNA polymerase 0.2 μ L；DdH2O13.8 μ L.

PCR reaction system is 20 μ L, amplification condition: 94 DEG C of denaturation 5min；94 DEG C of degeneration 30s, 59.7 DEG C of annealing 30s, 72 DEG C extend 60s, circulate 30 times；Last 72 DEG C extend 5min, and 4 DEG C terminate reaction.After having expanded, take PCR primer 3 μ L and detect with 0.8% agarose gel electrophoresis, and record result.

The abduction delivering of 2.3 recombination yeasts and the optimization of expression condition

The abduction delivering of (1) eight strain recombination yeast G-p-CoBoIFN-α

To identify that eight correct strain recombination yeasts are inoculated in 25mLBMGY culture medium respectively, and be placed in 30 DEG C of shaking tables and shake be cultured to OD600nm=2-6.Under room temperature, the centrifugal 5min of 2800r/min, collects thalline, is resuspended in BMMY, makes about OD600nm=1.0.Bacterium solution is placed in 1L shaking flask, 30 DEG C of 220rpm shaking tables are cultivated 96h.Every 24h adds 100% methanol to final concentration of 1.0% in culture medium.Bacterium solution is centrifuged, stays supernatant, be stored in-70 DEG C standby.

(2) eight strain recombination yeast G-p-CoBoIFN-α expressing quantities compare

1) Dot-ELISA (Dot-ELISA) of recombination yeast expressing quantity compares

Nitrocellulose filter is drawn as little lattice according to 13mm × 13mm unit, takes the supernatant after 10 μ L equivalent eight strains expression of recombinant yeast 96h containing CoBoIFN-α in corresponding lattice center, drying at room temperature.Film is put in the PBST solution containing 5% skimmed milk, close overnight for 4 DEG C.With PBST, film is washed 3 times, each 5min.Film is put in the BoIFN-α Anti-TNF-α liquid solution with PBST dilution (1:500), hatch 1h for 37 DEG C.With PBST, film is washed 3 times, each 5min.Film is put in the goat anti-rabbit igg of the PBST HRP labelling diluted (1:10000), hatch 1h for 37 DEG C.With PBST, film is washed 3 times, each 5min.Room temperature 4-CN Faxian color 10min.Put into and water terminates reaction, observed result.

Judged result: judge the expression height of eight strain recombination yeasts according to the depth with or without chromogenic reaction and colour developing.

2) indirect ELISA of recombination yeast expressing quantity compares

Take the supernatant after eight strain expression of recombinant proteins 96h, with the NaOH doubling dilution coated elisa plate of 0.01M.Primary antibodie is the BoIFN-α polyclonal antibody of 1:500 dilution, and negative control is not immune rabbit anteserum.Two goat anti-rabbit iggs resisting the HRP labelling for 1:10000 dilution.TMB color development at room temperature 15min.1M sulphuric acid terminates reaction, measures A value at microplate reader OD450nm place.

(3) optimization of recombination yeast G-p-CoBoIFN-α abduction delivering condition

Dominant strain activation Dot-ELISA and indirect ELISA identified, carries out abduction delivering, and concrete grammar is referring to 2.2.2.4.Optimize abduction delivering condition, use the baffle flask of 1000mL, make the volume of induction liquid and volume of a container ratio for about l:10, in shaking table, shake the supply cultivated to ensure oxygen with the speed of 220-250r/min；Temporally put 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h and take 3mL bacteria liquid sample respectively, collected after centrifugation supernatant, supernatant carries out SDS-PAGE analysis after TCA method concentrates, and the supernatant of expression is carried out indirect ELISA detection, analyze result with SDS-PAGE and indirect ELISA and determine the protein expression time of the best.

(4) abduction delivering of recombination yeast G-p-CoBoIFN-α dominant strain and G-p-Y-CoBoIFN-α and G-p-BoIFN-α G and recombiant protein is quantitative

According to the abduction delivering condition optimized, a large amount of abduction delivering recombination yeast G-p-CoBoIFN-α dominant strains and G-p-Y-CoBoIFN-α and G-p-BoIFN-α G, bacterium solution supernatant is collected in the best protein expression time, with the thick pure protein of saturated ammonium sulfate method, through the concentration of Bradford standard measure secretory protein after PBS, compare the expression of recombination yeast G-p-CoBoIFN-α dominant strain and G-p-Y-CoBoIFN-α and G-p-BoIFN-α G.Membrane filtration with 0.45 μm is degerming, detects for albumen antiviral activity.

(5) the indirect ELISA analysis of recombination yeast G-p-CoBoIFN-α dominant strain and G-p-Y-CoBoIFN-α and G-p-BoIFN-α G-protein expression

Take recombination yeast G-p-CoBoIFN-α dominant strain and G-p-Y-CoBoIFN-α and the NaOH doubling dilution coated elisa plate of G-p-BoIFN-α G96h expression supernatant 0.01M.Primary antibodie is the BoIFN-α polyclonal antibody of dilution 1:500, and negative control is not immune rabbit anteserum.Two goat anti-rabbit iggs resisting the HRP labelling for diluting 1:10000.TMB color development at room temperature 15min.1M sulphuric acid terminates reaction, measures A value at microplate reader OD450nm place.

(6) Westernblot of yeast secreted protein CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G analyzes

With reference to the Westernblot method in the third edition " Molecular Cloning: A Laboratory Methods Instruction " [51].According to a conventional method the yeast expression albumen after processing is carried out SDS-PAGE, be transferred on nitrocellulose filter, close overnight with 0.05g/mL skimmed milk 4 DEG C, PBST washes film 3 times, primary antibodie is rabbit anti-BoIFN-α A polyclonal antibody (1:500), and 37 DEG C of effects 1h, PBST wash film 3 times, two resist for HRP labelling goat anti-rabbit igg (1:10000), 37 DEG C of effects 1h, PBST wash film 3 times, and room temperature 4-CN method develops the color, put into and water terminates reaction, observed result.

3 results and analysis

Codon optimized and the synthesis of the design of 3.1CoBoIFN-α and CoBoIFN-α and BoIFN-α G

3.1.1CoBoIFN-the pcr amplification of α gene

(1) pcr amplification of fragment A, B, C, D

With pMD18-T-BoIFN-α-7 plasmid for template, Ye-BoIF5S and Aa2 is that primer carries out pcr amplification, it is thus achieved that the genetic fragment A of about 120bp；With pMD18-T-BoIFN-α-1 plasmid for template, Bs1 and Ba2 is that primer carries out pcr amplification, it is thus achieved that the genetic fragment B of about 180bp；With pMD18-T-BoIFN-α-6 plasmid for template, Cs1 and Ca2 is that primer carries out pcr amplification, it is thus achieved that the genetic fragment C of about 120bp；With pMD18-T-BoIFN-α-14 plasmid for template, Ds1 and Ye-BoIF3a is that primer carries out pcr amplification, it is thus achieved that the genetic fragment D of about 150bp；As it is shown on figure 3, be all consistent with intended purpose fragment A, B, C, D size.

(2) fusion of fragment A, B, C, D gene

Utilize overlap extension pcr, by fragment A, B, C, D gene fusion together, amplify the CoBoIFN-alpha gene fragment of about 570bp, be consistent with intended purpose clip size, as shown in Figure 4.

3.1.2Y-CoBoIFN-the gene optimization of α and Y-BoIFN-α G

Before and after sequence optimisation, each related data is such as shown in table 3-1, and as seen from table, by gene optimization, codon adaptability index has all brought up to 0.82, and G/C content is within the suitableeest scope, and mRNA free energy substantially reduces.As it is shown in figure 5, as seen from the figure, after gene optimization, utilization rate is 69% in the ratio shared by the codon of 91%-100% to the percent of the sub-utilization rate of each stream cipher, is significantly improved before relatively optimizing.

The each related data gene optimization result of table 3-1

3.1.3Y-CoBoIFN-the pcr amplification of α and Y-BoIFN-α G gene

Utilize overlap extension pcr, by 16 small fragment gene splicings together, amplify Y-CoBoIFN-α and the Y-BoIFN-α G genetic fragment of about 500bp, be consistent with intended purpose clip size, as shown in Figure 6.

The structure of 3.2 recombination yeasts containing CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α-G and expression

3.2.1 the structure of the Yeast expression carrier of CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α-G is contained

CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α-G gene after being reclaimed by double digestion purification connect in yeast expression vector pPICZ α A, construct recombinant plasmid pPICZ alpha A-CoBoIFN-α, pPICZ α A-Y-CoBoIFN-α, pPICZ α A-Y-BoIFN-α G, restricted enzyme XhoI is mono-cuts qualification, and XhoI and XbaI carries out double digestion qualification.Through 0.8% agarose gel electrophoresis, enzyme action result is analyzed: recombiant plasmid, through XhoI single endonuclease digestion, obtains the genetic fragment that size is about 4.2kb；Through XhoI and XbaI double digestion, obtain about 500bp size and the genetic fragment of about 3.6kb respectively, as shown in Figure 7.

3.2.2 the PCR of recombination yeast G-p-CoBoIFN-α, G-p-Y-CoBoIFN-α and G-p-Y-BoIFN-α G identifies

Boiling-to freeze-cooking method process after thalline supernatant for template, clone identification is carried out for primer pair CoBoIFN-α gene with 5 ' AOX1 and Ye-BoIF3a, clone identification is carried out for primer pair Y-CoBoIFN-α and Y-BoIFN-α G gene with Ye-BoIF5S, Ye-BoIF3a, product is through 0.8% agarose electrophoretic analysis, obtain about 800bp genetic fragment, it is consistent with intended purpose clip size, as shown in Figure 8.

3.2.3 the comparison of eight strain recombination yeast G-p-CoBoIFN-α expressing quantities

The Dot-ELISA (Dot-ELISA) of (1) eight plant weight group yeast protein expression analyzes

By the recombination yeast of eight strain pPICZ α A-CoBoIFN-α genes of random choose and pPICZ α A empty carrier recombination yeast abduction delivering, after expression, supernatant carries out Dot-ELISA, the relatively expression height of each strain recombination yeast, chooses the continuation downstream experiment that expression is the highest.The depth (see Fig. 9) according to immunodotting color is known: in eight strains recombination yeast containing pPICZ α A-CoBoIFN-α gene, the expression of No. 2 recombination yeasts is the highest, No. 5,9 numbers it, No. 1, No. 3, No. 7 relatively low, No. 4, No. 8 minimum.

The indirect ELISA analysis of (2) eight plant weight group yeast protein expressions

With indirect ELISA method, recombiant protein is detected, dilute recombiant protein coated elisa plate with 0.01MNaOH, BoIFN α polyvalent antibody 1:1000 dilution (negative control is not immune rabbit anteserum), the goat anti-rabbit igg 1:10000 dilution of HRP labelling.TMB develops the color, and 1M sulphuric acid terminates reaction, measures A value at microplate reader OD450nm place.Recombiant protein 1:1000 dilutes result and sees Figure 10.Result is consistent with Dot-ELISA result, it is determined that No. 2 recombination yeast CoBoIFN-alpha immunization reactivities are best.

3.2.4 the optimization of recombination yeast G-p-CoBoIFN-α abduction delivering condition

To the recombination yeast dominant strain containing CoBoIFN-α gene and pPICZ α A empty carrier recombinant yeast pichia pastoris abduction delivering simultaneously, 12%SDS-PAGE and indirect ELISA analysis is carried out, it has been found that secreted expressing quantity is the highest when inducing 96h after 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h time point takes the concentration of 3mL bacteria liquid sample respectively.As shown in FIG. 11 and 12.

3.2.5 the abduction delivering of recombination yeast G-p-CoBoIFN-α dominant strain and G-p-Y-CoBoIFN-α and G-p-Y-BoIFN-α G and recombiant protein is quantitative

According to the abduction delivering condition optimized, a large amount of abduction delivering recombination yeast G-p-CoBoIFN-α dominant strains and G-p-Y-CoBoIFN-α and G-p-BoIFN-α G, after after 96h, collection bacterium solution supernatant carries out preliminary purification and concentration, carry out SDS-PAGE analysis, can two protein bands visible in detail, size is 17kDa and 20kDa respectively, as shown in figure 13.Through Bradford standard measure, the concentration of recombinant protein c oBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G is respectively as follows: 0.382mg/mL, 0.579mg/mL, 0.564mg/mL.

3.2.6 the indirect ELISA analysis of recombination yeast G-p-CoBoIFN-α dominant strain and G-p-Y-CoBoIFN-α and G-p-Y-BoIFN-α G-protein expression

With indirect ELISA method, recombiant protein is detected, dilute recombiant protein coated elisa plate with 0.01MNaOH, BoIFN-α polyvalent antibody 1:500 dilution (negative control is not immune rabbit anteserum), the goat anti-rabbit igg 1:10000 dilution of HRP labelling.TMB develops the color, and 1M sulphuric acid terminates reaction, measures A value at microplate reader OD450nm place.Recombiant protein 1:1000 dilutes result and sees Figure 14.

3.2.7 the Westernblot of recombination yeast G-p-CoBoIFN-α dominant strain and G-p-Y-CoBoIFN-α and G-p-Y-BoIFN-α G expressing protein identifies

Westernblot result shows, the secretory protein of expression of recombinant yeast can with the goat anti-rabbit igg being primary antibodie horseradish peroxidase-labeled with rabbit anti-BoIFN-α A hypotype polyvalent antibody be two resist purpose band place occur specific chromogenic react, as shown in figure 15, it was demonstrated that obtain Niu Gongyou IFN-α and cattle IFN-α G subtype protein that correct secreting type optimizes.

The antiviral activity analysis of test example 1 yeast secreted protein CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G

The antiviral activity detection of 1 yeast secreted protein CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G

The preparation of 1.1VSV

(1) amplification of VSV and preservation

Press 1:1000 with the DMEM nutritional solution containing 10% top grade hyclone and dilute VSV seed culture of viruses, it is inoculated in well-grown monolayer bovine kidney cells (MDBK) to breed, when hatching to cell 80% pathological changes, multigelation 3 times, repeatedly blow and beat cell bottle wall with 10mL suction pipe, collect supernatant, be sub-packed in sterile centrifugation tube, frozen in-80 DEG C, seed culture of viruses is frozen in liquid nitrogen.

(2) the titer titration of VSV

MDBK cell monolayer is prepared in 96 orifice plates, discard nutritional solution, with the DMEM nutritional solution of serum-free by 10 times of multiple proportions serial dilution VSV viruses, each dilution factor does 6 cell repeating holes, every hole adds the virus liquid 100 μ L after dilution, cell negative control hole adds the DMEM nutritional solution of 100 μ L serum-frees, 37 DEG C of CO₂Hatching 1h, abandoning supernatant in constant incubator, every hole adds the DMEM nutritional solution 100 μ L containing 5% hyclone.When there is obvious pathological changes, cytopathy hole count in record cell hole, Karber method calculates the titer (TCID50) of VSV.

The mensuration of 1.2 antiviral activities

(1) cell is cultivated

When MDBK cell growth state is good, discard cell culture fluid, wash 2 times with 1 × PBS, after trypsinization, with the DMEM culture fluid containing 10% hyclone by cell piping and druming uniformly, it is distributed in 96 porocyte culture plates, then 96 porocyte plates are put into 37 DEG C of CO by every hole 100 μ L₂Constant incubator is cultivated, treats that Growth of Cells is to 60%.

(2) dilution and application of sample

With 4 times of doubling dilution interferon samples of the DMEM culture fluid containing 10% top grade hyclone, it is sub-packed in 1.5mL sterile centrifugation tube, the each hole of A1-H1 of 96 porocyte culture plates adds 10% top grade hyclone DMEM culture fluid 100 μ L, as noiseless element, virus-free cell control well, then in each hole of A2-H2, any process is not done, in order to the follow-up work virus control wells without interferon, the interferon diluted is added remaining 96 porocytes and cultivates in plate hole, every hole 100 μ L, each column is an interferon sample dilution.It is then placed in 37 DEG C of CO₂Constant incubator is cultivated 24h.

(3) counteracting toxic substances

Take the VSV virus measuring titer, be diluted to working concentration (100TCID50) with the DMEM nutritional solution of serum-free, take out step 2) the middle Tissue Culture Plate prepared, supernatant discarded, wash 2 times with 1 × PBS, add the virus liquid diluted, every hole 100 μ L, 37 DEG C of CO₂Hatching 1h, supernatant discarded in constant incubator, wash 2 times with 1 × PBS, every hole adds the DMEM nutritional solution 100 μ L containing 5% hyclone, puts into CO₂Constant incubator is cultivated, to the complete pathological changes of cell control well.

(4) dyeing

Discarding the supernatant in 96 porocyte culture plates in step 3), wash 3 times with 1 × PBS, every hole adds violet staining liquid 50 μ L, and room temperature places 20min, then washes 3 times with 1 × PBS again.Finally examine under a microscope cell death situation.

(5) result computational methods

Distance proportion=higher than the percent-50/ of 50% cytopathic effect inhibition is higher than the percent-lower than the percent of 50% cytopathic effect inhibition of 50% cytopathic effect inhibition, will be above the 50% dilution logarithm of cytopathic effect inhibition rate again to be added with distance proportion, result is exactly the logarithm (lg4) of the most high dilution of interferon 50% cytopathic effect inhibition rate.

The antiviral activity of 2 yeast secreted protein CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G blocks to be analyzed

MDBK cell monolayer is prepared in 96 orifice plates, anti-for rabbit after purification BoIFN-α polyclonal antibody 56 DEG C is inactivated 30min, filtration sterilization, after Growth of Cells is to 80%, after taking the CoBoIFN-α of 100U, Y-CoBoIFN-α and Y-BoIFN-α G and purification respectively, MDBK cell pellet overnight is hatched in the every hole of rabbit anti-BoIFN-alpha immunization serum 100 μ L of 2 times of doubling dilutions, sets nonimmune rabbit anteserum as negative control group simultaneously.VSV100 μ L every hole infection cell of 100TCID50,37 DEG C effect 1 hour after change complete culture solution, put into CO₂Constant temperature cell culture incubator is cultivated, after 24 hours, examines under a microscope cytopathy situation.

3 results and analysis

3.1 yeast secreted protein CoBoIFN-α, Y-CoBoIFN-α, Y-BoIFN-α G antiviral activity analysis

Utilize MDBK/VSV virus detection system, detect its antiviral activity by after 4 times of doubling dilutions of yeast secreted protein, and set empty carrier expressing protein matched group.Connecing after poison observation of cell pathological changes situation day by day, after meeting poison 24h, observation of cell has pathological changes in various degree, as shown in table 3-2,3-3,3-4 and Figure 16,17,18.Distance proportion=(percent-50% higher than 50% pathological changes rate)/(percent-lower than the percent of 50% pathological changes rate higher than 50% pathological changes rate), result calculates according to the observation, and yeast secreted protein CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G suppress the VSV activity replicated respectively [4 on MDBK cell^(9+0.226)×1]U/0.1mg/mL]/(0.04mg/mL)=8.98×10⁷U/mg、[4^(8+0.904)×1]U/0.1mg/mL]/(0.025mg/mL)=9.16×10⁷U/mg、[4^(9+0.4)×1]U/0.1mg/mL]/(0.05mg/mL)=9.12×10⁷U/mg。

Table 3-2CoBoIFN-α antiviral activity detection on MDBK cell

Table 3-3Y-CoBoIFN-α antiviral activity detection on MDBK cell

Table 3-4Y-BoIFN-α G antiviral activity detection on MDBK cell

3.2 yeast secreted protein CoBoIFN-α, Y-CoBoIFN-α, Y-BoIFN-α G antiviral activity block analyze

Antiviral activity blocks result and shows, the antiviral activity of CoBoIFN-α, Y-CoBoIFN-α and Y-BoIFN-α G effectively can be blocked by the anti-BoIFN-alpha immunization polyvalent antibody of rabbit, suppresses the VSV ability replicated on MDBK thus losing.Effective extension rate of the CoBoIFN-α that rabbit anti-BoIFN-alpha immunization polyvalent antibody blocks 100U yeast secreted expression completely is 128, effective extension rate of Y-CoBoIFN-α and the Y-BoIFN-α G-protein blocking 100U yeast secreted expression completely is 64, nonimmune rabbit negative serum then can not block the antiviral activity of this albumen completely, as shown in figure 19.

Claims (10)

1. recombinant bovine alpha interferon CoBoIFN-α, it is characterised in that the aminoacid sequence of interferon CoBoIFN-α is shown in SEQIDNo.32.

2. the nucleotide sequence of coding recombinant bovine alpha interferon CoBoIFN-α described in claim 1, it is characterised in that described nucleotides sequence is classified as shown in SEQIDNo.31.

3. an expression vector, it is characterised in that containing the nucleotide sequence described in claim 2.

4. a host cell, it is characterised in that containing the carrier described in claim 3.

5. the application in the medicine of preparation treatment or prevention cattle disease viral disease of the recombinant bovine alpha interferon CoBoIFN-α described in claim 1, the virus causing described cattle disease viral disease is vesicular stomatitis virus.

6. recombinant bovine alpha interferon Y-CoBoIFN-α, it is characterised in that the aminoacid sequence of interferon Y-CoBoIFN-α is shown in SEQIDNo.34.

7. the nucleotide sequence of coding recombinant bovine alpha interferon Y-CoBoIFN-α described in claim 6, it is characterised in that described nucleotides sequence is classified as shown in SEQIDNo.33.

8. an expression vector, it is characterised in that containing the nucleotide sequence described in claim 7.

9. a host cell, it is characterised in that containing the carrier described in claim 8.

10. the application in the medicine of preparation treatment or prevention cattle disease viral disease of the recombinant bovine alpha interferon Y-CoBoIFN-α described in claim 6, the virus causing described cattle disease viral disease is vesicular stomatitis virus.