CN102533797A - Preparation method for recombinant coxsackie virus A16 like particle and applications thereof - Google Patents

Abstract

The invention discloses a preparation method for a recombinant coxsackie virus A16 like particle, which comprises the following steps: (1) cloning a P1 gene and a 3CD gene of a coxsackie virus A16 to a target plasmid to obtain a recombinant expression vector; (2) transforming a target yeast cell by using the recombinant expression vector obtained in the step (1) to obtain a recombinant yeast cell for expressing the P1 gene and the 3CD gene; and (3) cracking the recombinant yeast cell obtained in the step (2), and separating to obtain the recombinant coxsackie virus A16 like particle. The recombinant coxsackie virus A16 like particle can be prepared in a yeast expression system by the method provided by the invention. Compared with a wild-type P1 gene and a wild-type 3CD gene, the yield of the coxsackie virus A16 like particle in the yeast expression system is greatly increased through the optimization of codons of the P1 gene and the 3CD gene, and the recombinant coxsackie virus A16 like particle can be further used for producing candidate preventive vaccines and pharmaceutical compositions for infant hand-foot-and-mouth diseases.

Description

The preparation method and the application thereof of reorganization coxsackie virus A 16-type virus-like particle

Technical field

The preparation method of coxsackie virus A 16-type virus-like particle the present invention relates to recombinate; Be particularly related to the coxsackie virus A 16-type P1 gene and the 3CD gene that utilize codon to optimize, prepare the method for reorganization coxsackie virus A 16-type virus-like particle through yeast expression system.

Background technology

(Hand foot mouth disease HFMD) is the common transmissible disease of infant to hand foot mouth disease, and by Coxsackie virus (Coxsackie virus) A group 16,4,5,7,9,10 types that the HEV belongs to, B organizes 2,5 types; Echo virus (ECHO viruses) and enterovirns type 71 (EV 71) infect and cause.Wherein with EV 71 and the most common (the Schmidt NJ of Cox A16 type; Lennette EH, Ho HH.An apparently new enterovirus isolated from patients with disease of the central nervous system.J Infect Dis 1974; 129:304-309.).The CoxA16 infection can cause complication such as myocarditis, myocardosis and aseptic meningitis; In addition, CoxA16 still is the pathogenic former of multiple human diseases, can cause (Yang F such as respiratory tract infection, conjunctivitis, heat generation fash, herpangina, acute flaccid paralysis; Zhang T; Hu YF.Survey of Enterovirus Infections from Hand, Footand Mouth Disease Outbreak in China, 2009.Virology Journal 2011; 8:1-4.).End on October 31,2008 1 day to 2011 January in, and nearly 4,760,000 examples of hand foot mouth disease are infected in the whole nation, cause 1821 infant death.In the hand foot mouth disease of China's outburst in 2009, find, have the case that infects EV71 and two kinds of viruses of CoxA16 simultaneously.

Coxsackie virus A 16-type (Coxsackievirus A16 CoxA16) belongs to picornavirus, and its virion is the icosahedro bodily form, three-dimensional symmetry, and shape spherical in shape is the nucleocapsid that exposes, diameter is about 23-30nm, no coating.Viral genome is the single stranded RNA of about 7kb, and it is formed from 5 ' and holds the direction of 3 ' end to be followed successively by 5 ' end non-coding region, and P1 district, P2 district, P3 district and one section 3 ' are held non-coding region.The primary structure albumen of P1 district coding virus, i.e. capsid protein (capsule protein), its transcribe and translation product behind protease hydrolysis, finally obtain 4 protein products, be respectively VP4 (1A), VP2 (1B), VP3 (1C) and VP4 (1D).In main with antigen site just in the P1 district on the VP1, also contain in some on VP2 and the VP3 and the antigenic determinant of type, but the zone of their existence is in non-conserved sequence.The VP4 sequence is high conservative, is not exposed to the outside surface of capsid, so do not have the neutrality antigenic determinant.The P2 district presents P2A, P2B and three parts of P2C.Proteic this zone of P2A has been to suppress host cell mRNA Transcription.The function of P2B is with to change membrane interaction relevant, and P2C albumen it participates in that viral RNA is genomic to be duplicated by inference.In the P3 district, VPg (P3A) precursor is through being processed into sophisticated VPg.P3C is a proteolytic enzyme, can the autocatalytically oneself shear release.3D is a RNA synthetic enzyme, is the RNA polymerase of the dependenc RNA in the rna replicon process, and it can duplicate the RNA of synthetic virus.

Research shows that CoxA16 inactivation of viruses immune mouse can produce anti-CoxA16 and the two kinds of neutralizing antibodies of anti-EV71 with protection effectiveness; Can significantly improve survival rate (the Wu TC of laboratory animal in the challenge test; Wang YF, Lee YP.Immunity to Avirulent Enterovirus 71 and Coxsackie A16 Virus Protects against Enterovirus 71 Infection in Mice.Journal Of Virology 2007; 81:10310-10315.).But at present, specifics or the vaccine listing also do not infected to CoxA16, therefore, development CoxA16 preventative vaccine is popular significant for control infant hand foot mouth disease.And virus-like particle on form with real virus type seemingly, and do not contain potential Causative virus genome, so virus-like particle is the exploitation of antiviral preventative vaccine the ideal alternatives is provided.

Summary of the invention

An object of the present invention is to provide a kind of preparation method of coxsackie virus A 16-type virus-like particle.

The preparation method of coxsackie virus A 16-type virus-like particle provided by the present invention comprises the steps:

1) with the P1 gene of coxsackie virus A 16-type and 3CD gene clone in the purpose plasmid, obtain recombinant expression vector;

2) recombinant expression vector that obtains with step 1) transforms the purpose yeast cell, obtains expressing the recombinant yeast cell of said P1 gene and 3CD gene;

3) cleavage step 2) recombinant yeast cell that obtains, separate the coxsackie virus A 16-type virus-like particle that obtains recombinating.

Above-mentioned steps 1) in, said P1 gene and said 3CD gene are the gene that codon is optimized; Said being optimized under the prerequisite that does not change wild-type coxsackie virus A 16-type P1 albumen and wild-type coxsackie virus A 16-type 3CD Argine Monohydrochloride sequence replaces with the codon of wild-type coxsackie virus A 16-type P1 gene and wild-type coxsackie virus A 16-type 3CD gene the codon of yeast cell preference (high frequency use).Said optimization also comprises other transformations to P1 gene order and 3CD gene order, expresses in the yeast cell being adapted at.The term that uses among the present invention " codon of preference " has implication well known in the art; Be also referred to as the preference property (CodonPreference) of codon, be meant that some organism is more had a preference for to use some synonym codeword triplet (codon of the same amino acid of promptly encoding).

The nucleotides sequence of P1 gene in one embodiment of the invention, above-mentioned steps 1) (gene that codon is optimized, called after CoxA16P1Y) is classified as in the sequence table shown in the sequence 1; The nucleotides sequence of said 3CD gene (gene that codon is optimized, called after Cox A163CDY) is classified as in the sequence table shown in the sequence 2.For being adapted at expressing in the yeast cell, other gene orders that the method for describing according to the present invention is transformed acquisition to the P1 gene or the 3CD gene of coxsackie virus A 16-type also belong to protection scope of the present invention.

Above-mentioned steps 2) in, said purpose yeast cell can be yeast saccharomyces cerevisiae, multiple-shaped nuohan inferior yeast, pichia pastoris phaff, cell wall Crewe Vickers yeast, newborn Crewe Vickers yeast or grain wine fragmentation sugar yeast.In one embodiment of the invention, said purpose yeast cell is specially yeast saccharomyces cerevisiae INVSC1.

Each is transcribed by a promoters driven in said recombinant expression vector for said P1 gene and 3CD gene, and the promotor direction that drives said P1 gene is in the opposite direction with the promotor that drives said 3CD gene.In one embodiment of the invention, said recombinant expression vector is for being inserted into said P1 gene and said 3CD gene respectively two MCS places of pESC-URA.Specifically can said P1 gene be inserted between the SpeI and Pac I of MCS 1 (MCS1/FLAG) of said pESC-URA; Said 3CD gene is inserted between the Sal I and Kpn I of MCS 2 (MCS2/myc) of said pESC-URA the recombinant plasmid that obtains.

The coxsackie virus A 16-type virus-like particle that utilizes method provided by the present invention to prepare also belongs to protection scope of the present invention.

Another object of the present invention provides the P1 gene and the 3CD gene of the coxsackie virus A 16-type of codon optimized type.

The nucleotides sequence of the P1 gene of the coxsackie virus A 16-type of codon optimized type provided by the present invention (CoxA16 P1Y) is classified as in the sequence table shown in the sequence 1; The nucleotides sequence of the 3CD gene of the coxsackie virus A 16-type of codon optimized type provided by the present invention (Cox A16 3CDY) is classified as in the sequence table shown in the sequence 2.For being adapted at expressing in the yeast cell, other gene orders that the method for describing according to the present invention is transformed acquisition to the P1 gene or the 3CD gene of coxsackie virus A 16-type also belong to protection scope of the present invention.

Wherein, sequence 1 is made up of 2589 Nucleotide, and for optimizing the encoding sequence of back coxsackie virus A 16-type P1 gene, the albumen of its coding gained is the albumen shown in the sequence 5 in the sequence table, and sequence 5 is made up of 862 amino acid; Sequence 2 is made up of 1941 Nucleotide, is the encoding sequence of the coxsackie virus A 16-type 3CD gene after optimizing, and the albumen of its coding gained is the albumen shown in the sequence 6 in the sequence table, and sequence 6 is made up of 646 amino acid.

Recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium of P1 gene or 3CD gene that contains the coxsackie virus A 16-type of above-mentioned codon optimized type also belongs to protection scope of the present invention.

Said recombinant expression vector can be two MCS places that said P1 gene and said 3CD gene are inserted into the pESC-URA plasmid respectively, the recombinant plasmid that obtains.Said reorganization bacterium can be the P1 gene of the coxsackie virus A 16-type that carries above-mentioned codon optimized type or the recombinant yeast cell of 3CD gene.Said yeast cell can be yeast saccharomyces cerevisiae, multiple-shaped nuohan inferior yeast, pichia pastoris phaff, cell wall Crewe Vickers yeast, newborn Crewe Vickers yeast or grain wine fragmentation sugar yeast.In one embodiment of the invention, said yeast cell is specially yeast saccharomyces cerevisiae INVSC1.

Said expression cassette is by the P1 gene of the coxsackie virus A 16-type that can start said codon optimized type or the promotor of 3CD genetic expression, the P1 gene of the coxsackie virus A 16-type of said codon optimized type or 3CD gene, and transcription terminator is formed.In one embodiment of the invention; Said promotor is specially yeast saccharomyces cerevisiae GAL1 and GAL10 promotor; Can certainly use any of other known Yeast promoters, like GAL7, ADH1, ADH3 and PGK promotor, perhaps other eukaryotic gene promotors.In one embodiment of the invention, said transcription terminator is specially ADH1 and CYC1 terminator.

The P1 gene of the coxsackie virus A 16-type of above-mentioned codon optimized type or 3CD gene the preparation following a) or b) in application also belong to protection scope of the present invention:

A) coxsackie virus A 16-type virus-like particle;

B) with the coxsackie virus A 16-type virus-like particle be the preventative vaccine of activeconstituents.

The invention still further relates to and provide and in recombinant host cell, express Cox A16 P1 albumen and the proteic method of 3CD simultaneously; This method comprises: the recombinant expression vector that coding Cox A16 P1 albumen and the proteic encoding sox of 3CD will be contained in (1) imports recombinant host cell, like the yeast cell (yeast saccharomyces cerevisiae INVSC1) of reorganization.(2) under the condition that allows described Cox A16 P1 albumen and 3CD albumen to express simultaneously, cultivate recombinant host cell, like the yeast cell (yeast saccharomyces cerevisiae INVSC1) of reorganization.

The present invention is to coxsackie virus A 16-type (the Coxsackievirus A16 of wild-type; CoxA16) it is codon optimized that P1 gene or 3CD gene have carried out yeast cell preference type, thereby improved CoxA16 P1 albumen and the expression level (see figure 5) of CoxA16 3CD albumen in yeast cell.In the present invention; Cox A16 P1 albumen and CoxA16 3CD albumen are expressed in yeast cell simultaneously; Cox A16 3CD albumen to Cox A16P1 albumen carry out obtaining after enzyme is cut VP1, VP2, VP3, (3CD is the precursor of 3C to four kinds of structural protein of VP4; The 3C albumen that is produced by 3CD albumen is a kind of proteolytic enzyme, can be VP1, VP2, VP3, four kinds of structural protein of VP4 with the P1 proteolyze).This four kinds of structural protein and then be assembled into the virus-like particle (Cox A16VLPs) of coxsackie virus A 16-type voluntarily.

Experiment showed, the method provided by the present invention of utilizing, in yeast expression system (yeast saccharomyces cerevisiae INVSC1) successful preparation Cox A16 VLPs.Compare with the 3CD gene than wild-type P1 gene simultaneously, the present invention is to the codon optimized output of Cox A16 VLPs in yeast expression system that improved greatly of P1 gene and 3CD gene.Said Cox A16 VLPs can be used to prepare the Cox A16 preventative vaccine based on VLP.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure of pESC-P1Y plasmid.Wherein, use swimming lane A to be 15000bp DNA Marker; Swimming lane B is the contrast of pESC-URA plasmid; Swimming lane C is the pESC-P1Y plasmid.

Fig. 2 cuts qualification result for the SpeI and the Pac I enzyme of pESC-P1Y plasmid.Wherein, use swimming lane A to be 15000bpDNA Marker; Swimming lane B is that the SpeI and the Pac I enzyme of pESC-P1Y plasmid cut qualification result.

Fig. 3 is the agarose gel electrophoresis figure of pESC-P1Y-3CDY plasmid.Wherein, use swimming lane A to be 15000bpDNA Marker; Swimming lane B is the contrast of pESC-P1Y plasmid; Swimming lane C and swimming lane D are the pESC-P1Y-3CDY plasmid.

Fig. 4 cuts qualification result for the Sal I and the Kpn I enzyme of pESC-P1Y-3CDY plasmid.Wherein, use swimming lane A to be 15000bp DNA Marker; Swimming lane B is that the Sal I and the Kpn I enzyme of pESC-P1Y-3CDY plasmid cut qualification result.

Fig. 5 identifies the expression of recombinant C ox A16 capsid protein for Western Blot.Wherein, swimming lane A is the molecular weight of albumen contrast; Swimming lane B is the INVSC1/pESC-P1Y-3CDY protein extract; Swimming lane C is an INVSC1 Yeast protein extract; Swimming lane D is the INVSC1/pESC-P1W-3CDW protein extract.

Fig. 6 is the representative sample of the Cox A16 virus-like particle (VLPs) that manifests through transmission electron microscopy.

Embodiment

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

Codon optimized and the full gene of embodiment 1, Cox A16 P1 gene and Cox A16 3CD gene is synthetic

Below by the codon optimized and full gene synthetic flow process of describing Cox A16 P1 gene and Cox A16 3CD gene step by step in detail.

1, Cox A16 P1 gene and Cox A16 3CD gene is codon optimized

According to wild-type Cox A16 P1 gene order (shown in sequence in the sequence table 3) and wild-type Cox A16 3CD gene order (shown in sequence in the sequence table 4); Obtain the optimization type Cox A16 P1 gene order and the optimization type Cox A16 3CD gene order that are suitable in yeast cell, expressing through design and repeated validation; The P1 gene order and the 3CD gene order that are about to Cox A16 virus are transformed under the prerequisite that does not change aminoacid sequence and contain just like (Sharp PM such as Sharp PM; Cowe E.Synonymous Codon Usage inSaccharomyces cerevisiae.Yeast; 1991; 7 (7): the 657-678.) majorizing sequence of the preferred codon of described yeast, thus improve Cox A16 P1 albumen and the expression level of Cox A16 3CD albumen in the yeast cell culture environment.

According to aforesaid method, final optimization type Cox A16 P1 gene and the Cox A16 3CD gene that obtains to press the design of yeast codon preference, difference called after Cox A16 P1Y and Cox A16 3CDY, its gene order is respectively sequence 1 and sequence 2 in the sequence table.Cox A16 P1Y gene (sequence 1) encoded protein is consistent with the albumen of the A16 of wild-type Cox shown in the sequence 3 P1 genes encoding, is the albumen that aminoacid sequence shown in the sequence 5 is formed in the sequence table; Cox A16 3CDY gene (sequence 2) encoded protein is consistent with the albumen of the A16 of wild-type Cox shown in the sequence 4 3CD genes encoding, is the albumen that aminoacid sequence shown in the sequence 6 is formed in the sequence table.

2, the full gene of codon optimized type Cox A16 P1 gene and Cox A16 3CD gene is synthetic

Use synthetic Cox A16 P1Y of the full gene of the overlapping extension PCR method of sequence and Cox A16 3CDY; Concrete grammar is following: according to the upstream and downstream fragment of synthetic many 100bp of the gene order of optimizing Cox A16 P1Y and Cox A16 3CDY, and two complementary overlapping 15bp of intersegmental 3 ' end, said upstream and downstream fragment template, primer is each other carried out pcr amplification; Use gel to reclaim test kit and reclaim amplified fragments; Again with this adjacent recovery fragment each other primer, template carry out the next round pcr amplification, obtain splicing sequence, template, primer carry out PCR splicing each other with the contiguous concatenation sequence again; Reclaim the splicing sequence fragment; Template, primer carry out the PCR splicing, the codon optimized type Cox A16 P1 gene and the Cox A163CD gene of final synthetic total length that the rest may be inferred, i.e. Cox A16 P1Y and Cox A163CD Y each other in sequence according to this.

Embodiment 2, carry the structure of the yeast recombinant expression vector of Cox A16 P1Y and Cox A16 3CD Y

1, the preparation of bacillus coli DH 5 alpha competent cell

The single bacterium colony (Takara company, catalog number is D9057S) of picking DH5 α from 37 ℃ of fresh flat boards of cultivating 16～20h is inoculated in 5ml and does not contain in the LB substratum of microbiotic 37 ℃ of thermal agitation overnight cultures (12～16h).From above-mentioned culture, draw 0.5ml next day and changed the about 3h of continuation cultivation in the 50ml LB substratum in 1: 100 by volume over to; OD600 value to bacterium liquid is 3 o'clock; Under aseptic condition, bacterium is transferred in aseptic, the ice-cold 50ml centrifuge tube ice bath 30min.Under 4 ℃ of conditions, the centrifugal 10min of 4000rpm abandons supernatant, will manage and be inverted 1min, residual nutrient solution is flow to end, with the resuspended bacterial precipitation of CaCl2 solution of the ice-cold 100mM of 10ml, ice bath 30min.4 ℃, the centrifugal 10min of 4000rpm abandons supernatant, and every 50ml initial incubation thing is with the CaCl of the 100mM that contains 15% glycerine of 2ml precooling ₂The resuspended bacterial precipitation of solution is distributed into the every pipe of 200 μ l, and-80 ℃ of preservations are subsequent use.

2, the conversion of pESC-URA plasmid

Draw 1 μ g pESC-URA plasmid (a kind of expression plasmid of yeast respectively with aseptic suction nozzle with bidirectional promoter; Available from Angilent technologies; Catalog number is 217454) add in the DH5 α competent cell of 200 μ l above-mentioned steps, 1 preparation; Rotate mixing gently, ice bath 30min.To manage and move into water-bath 90s in 42 ℃ of water baths, will manage then and transfer to fast in the ice bath, make cell cooling 1～2min.Every pipe adds 800 μ l and does not contain antibiotic LB substratum (peptone, yeast extract, penbritin are glad through company of section available from Beijing; NaCl is available from traditional Chinese medicines chemical reagent ltd); Pipe is transferred on 37 ℃ of shaking tables, and incubation 45min (rotating speed＜150rpm).Get the competent cell that 50 μ l have transformed, be coated onto gently with an aseptic elbow glass rod and contain corresponding antibiotic LB planar surface, place room temperature to liquid to be absorbed flat board, be inverted plate, cultivate 12～16h in 37 ℃.

3, a small amount of of pESC-URA plasmid preparation

Picking transforms single colony inoculation of cultivating gained through above-mentioned steps 2 and contains in the LB substratum of penbritin 37 ℃ of thermal agitation overnight cultures in 5ml.Culture is moved in the 1.5ml Eppendorf pipe, and the centrifugal 30-60s of 12000rpm removes nutrient solution; Be inverted centrifuge tube, supernatant is flow to end, (final concentration is the glucose of 50mmol/L with the solution I of 100 μ l precoolings; Final concentration is the Tris-HCl of 25mmol/L; Final concentration is the EDTA of 10mmol/L, pH8.0) resuspended thalline, thermal agitation mixing; Add the freshly prepared solution II of 200 μ l (final concentration is the NaOH of 0.2mol/L, and final concentration is the SDS of quality percentage composition 1%), gentle mixing, ice bath 3min, limpid to liquid; The gentle mixing of solution III (preparation 100ml solution III: 5mol/L KAc 60ml, glacial acetic acid 11.5ml, water 28.5ml) that adds 150 μ l precoolings; Ice bath 10min, the centrifugal 10min of 12000rpm carefully draws supernatant; Supernatant is transferred in another centrifuge tube, adds the absolute ethyl alcohol precipitation at room temperature 30min of 2 times of volumes, the centrifugal 10min of 12000rpm; Abandon supernatant, deposition is washed once with 70% ethanol, after room temperature is dried; Contain TE (pH 8.0) dissolution precipitation of RNaseA (100 μ g/ml) with 30 μ l, 37 ℃ of rearmounted-20 ℃ of preservations of water-bath 30～60min are subsequent use.

4, the structure of yeast recombinant expression vector pESC-P1Y

The Cox A16 P1Y gene clone that embodiment 1 is obtained obtains yeast recombinant expression vector pESC-P1Y between the SpeI and PacI of the MCS 1 (MCS1/FLAG) of pESC-URA plasmid.Specifically carry out according to following steps:

5 ' end of the Cox A16 P1Y gene (shown in sequence in the sequence table 1) that 1) embodiment 1 is obtained is introduced the restriction enzyme site of restriction enzyme SpeI and Pac I (restriction enzyme SpeI and Pac I are available from the precious biotechnology in Dalian ltd) respectively with 3 ' end, holds the restriction enzyme site of introducing restriction enzyme Sal I and Kpn I (restriction enzyme Sal I and Kpn I are available from Dalian treasured biotechnology ltd) respectively at the 5 ' end and 3 ' of Cox A16 3CD Y gene (shown in sequence in the sequence table 2).Concrete operations are following: (1) is template with the Cox A16 P1Y gene (shown in sequence in the sequence table 1) that embodiment 1 obtains, and carry out pcr amplification with following primer and obtain 5 ' end and hold the Cox A16P1Y gene fragment introduce restriction enzyme SpeI and Pac I restriction enzyme site respectively with 3 ': upstream primer is 5 '-GG ACTAGTACCATGGGTTCTCAAGTTTCTACTCA-3 ' (the underscore place is the sequence of restriction enzyme SpeI restriction enzyme site); Downstream primer is 5 '-CC TTAATTAATTACAAAGTAGTAATTTTATAGAA-3 ' (the underscore place is the sequence of restriction enzyme Pac I restriction enzyme site).(2) the Cox A16 3CD Y gene (shown in sequence in the sequence table 1) that obtains with embodiment 1 is a template, and carry out pcr amplification with following primer and obtain 5 ' end and hold the Cox A16 3CD Y gene fragment introduce restriction enzyme Sal I and Kpn I restriction enzyme site respectively with 3 ': upstream primer is 5 '-ACGC GTCGACACCATGGGT-3 ' (the underscore place is the sequence of restriction enzyme Sal I restriction enzyme site); Downstream primer is 5 '-GG GGTACCTTAAAACAATTCCAACCAATTTCT-3 ' (the underscore place is the sequence of restriction enzyme Kpn I restriction enzyme site).

2) use restriction enzyme SpeI and Pac I enzyme to cut the pESC-URA plasmid of above-mentioned steps 3 preparations; Reclaim skeleton carrier (big fragment; Because the small segment size only is about 45bp; Often can't show during agarose gel electrophoresis) with the Cox A16 P1Y gene fragment of cutting through same enzyme by 1: 4 mixed, add 10 * T4 dna ligase damping fluid and T4 dna ligase (T4 dna ligase and 10 * T4 dna ligase damping fluid are available from Dalian treasured biotechnology ltd) successively, reaction volume is 20 μ l; 16 ℃ of connections are spent the night, and get 10 μ l ligation product transformed into escherichia coli DH5 α competent cells.From the inoculation converted product the LB agar plate on several single bacterium colonies of picking, be inoculated into 5ml and contain in the corresponding antibiotic LB substratum, 37 ℃ of thermal agitation overnight cultures, the quick a small amount of preparation method of 3 said plasmids extracts recombinant plasmid (Fig. 1) set by step.And with restriction endonuclease SpeI and Pac I recombinant plasmid is carried out enzyme and cut evaluation; Qualification result is as shown in Figure 2; Obtain two bands that size is about 6600bp and 2600bp; Conform to expected results, further the recombinant plasmid after enzyme is cut evaluation send company's order-checking, order-checking is confirmed to contain the recombinant plasmid called after pESC-P1Y of Cox A16 P1Y gene.Adopt identical method to make up and contain wild-type Cox A16 P1 gene (Cox A16 P1W, sequence 3) contrast recombinant plasmid, called after pESC-P1W.

5, the structure of yeast recombinant expression vector pESC-P1Y-3CDY

The MCS 2 of the yeast recombinant expression vector pESC-P1Y that the said Cox A16 3CDY gene clone of embodiment 1 preparation is prepared to above-mentioned steps 4 (is the MCS 2 of pESC-URA plasmid; MCS2/myc) between the Sal I and Kpn I, obtain yeast recombinant expression vector pESC-P1Y-3CDY.Specifically carry out according to following steps:

Use restriction enzyme Sal I and Kpn I enzyme to cut the yeast recombinant expression vector pESC-P1Y of above-mentioned steps 4 preparations; The CoxA16 3CDY gene fragment that recovery skeleton carrier (big fragment) and the same enzyme of warp are cut was by 1: 4 mixed (DNA Marker and DNA sepharose recovery test kit are available from the precious biotechnology in Dalian ltd); Add 10 * T4DNA ligase enzyme damping fluid and T4DNA ligase enzyme successively; Reaction volume is 20 μ l; 16 ℃ of connections are spent the night, and get 10 μ l ligation product transformed into escherichia coli DH5 α competent cells.From the inoculation converted product the LB agar plate on several single bacterium colonies of picking, be inoculated into 5ml and contain in the corresponding antibiotic LB substratum, 37 ℃ of thermal agitation overnight cultures, the quick a small amount of preparation method of 3 said plasmids extracts recombinant plasmid (Fig. 3) set by step.And with restriction endonuclease Sal I and Kpn I recombinant plasmid is carried out enzyme and cut evaluation; Qualification result is as shown in Figure 4; Obtain two bands that size is about 9200bp and 1900bp; Conform to expected results, further the recombinant plasmid after enzyme is cut evaluation send company's order-checking, order-checking is confirmed to contain the recombinant plasmid called after pESC-P1Y-3CDY of CoxA16 3CDY gene.Among the yeast recombinant expression vector pESC-P1Y-3CDY, the promotor that starts said CoxA16 P1Y genetic transcription is a yeast saccharomyces cerevisiae GAL10 promotor, and transcription terminator is the ADH1 terminator; The promotor that starts said CoxA16 3CDY genetic transcription is a yeast saccharomyces cerevisiae GAL1 promotor, and transcription terminator is the CYC1 terminator.Adopt identical method to make up the contrast recombinant plasmid that contains wild-type CoxA16 P1 gene (CoxA16 P1W, sequence 3) and wild-type CoxA16 3CD gene (CoxA16 3CDW, sequence 4) simultaneously, called after pESC-P1W-3CDW.

Embodiment 3, recombinant C oxA16 virus-like particle great expression and the purifying in yeast cell

1, the preparation of yeast saccharomyces cerevisiae competent cell

Picking mono-clonal bacterium colony INVSc 1 (Saccharomyces cerevisiae, catalog number: C81000 is available from Invitrogen) is inoculated in the 10ml YPD nutrient solution (glad through company of section available from Beijing) in the yeast saccharomyces cerevisiae flat board; 16h is cultivated in 30 ℃ of joltings, and the culture of getting suitable volumes adds mixing in the 48ml YPD nutrient solution, and making its OD600 value is 0.5; The OD600 value is 0.7 behind 30 ℃ of joltings cultivation 1h, after continuing to cultivate 30min, culture is changed in the aseptic centrifuge tube of 50ml; The centrifugal 5min of room temperature 1500rpm; Abandon supernatant, with the resuspended deposition of solution I (test kit carries) among the 10ml Sc EasyComp transformation kit (available from Invitrogen), the centrifugal 5min of room temperature 1500rpm; Carefully abandon supernatant; With the resuspended deposition of 1ml solution II (test kit carries), in the 1.5ml centrifuge tube of 50 μ l/ pipe packing sterilization, put-70 ℃ of preservations.

2, yeast recombinant expression vector pESC-P1Y-3CDY transformed yeast competent cell

The yeast recombinant expression vector pESC-P1Y-3CDY of 4 μ l embodiment, 2 preparations is joined in the yeast INVSC1 competent cell of above-mentioned steps 1 preparation; Add the solution III among the 500 μ l Sc EasyComp transformation kit (available from Invitrogen); Thermal agitation mixing on the vortex vibrator obtains the DNA/ yeast mixt.The DNA/ yeast mixt put in 30 ℃ of water-baths hatch 1h, every at a distance from 15min thermal agitation mixing on the vortex vibrator.Get 100 μ l coating URA and select dull and stereotyped (selectivity nutrient solution (Ura-): YNB6.7g; Yeast Ura auxotrophy substratum powder (Sigma company, Y1501) 1.92g, as make the dull and stereotyped 20g of adding agar powder; Be dissolved in the 900ml deionized water; Autoclaving is cooled to 50 ℃, adds 20% aseptic glucose of 100ml.) 0-, put to be inverted in 30 ℃ of incubators and cultivated 2-4 days.5 mono-clonal colony inoculations of picking are selected in the nutrient solution in 10ml URA at random, and 16h, centrifugal collection yeast cell are cultivated in 30 ℃ of joltings.To pass through above-mentioned evaluation and show that success changes the yeast INVSC1 cell called after INVSC1/pESC-P1Y-3CDY of recombinant expression vector pESC-P1Y-3CDY over to.The contrast of the yeast INVSC1 cell that changes the pESC-URA empty carrier over to is set simultaneously, with this yeast cell called after INVSC1/pESC-URA.Adopt identical method to obtain changing over to the yeast INVSC1 cell of recombinant expression vector pESC-P1W-3CDW, with its called after INVSC1/pESC-P1W-3CDW.

3, great expression and the purifying of recombinant C ox A16 virus-like particle in yeast cell

1) great expression of recombinant C ox A16 virus-like particle in yeast cell: INVSC1/pESC-P1Y-3CDY (experimental group), INVSC1/pESC-P1W-3CDW (control group) and the single bacterium colony of INVSC1/pESC-URA (negative control group) that will pass through above-mentioned steps 2 acquisitions are inoculated in 50ml URA respectively and select nutrient solution (selectivity nutrient solution (Ura-): YNB 6.7g; Yeast Ura auxotrophy substratum powder (Sigma company; Y1501) 1.92g; Be dissolved in the 900ml deionized water; Autoclaving is cooled to 50 ℃, adds 20% aseptic glucose of 100ml.) in, 16h is cultivated in 30 ℃ of joltings, and the nutrient solution of getting suitable volumes is inoculated in 500ml URA and selects in the nutrient solution, and making the OD600 value is 0.4; 4h is cultivated in 30 ℃ of joltings, gets nutrient solution inoculation 3L URA inducing culture liquid (YNB6.7g, yeast Ura auxotrophy substratum powder (Sigma company, Y1501) 1.92g of suitable volumes; Be dissolved in the 800ml deionized water, autoclaving is cooled to 50 ℃; Add aseptic 20% semi-lactosi and the 20% aseptic SANMALT-S of 100ml of 100ml), making the OD600 value is that 48h is cultivated in 0.4,30 ℃ of jolting; Centrifugal collection bacterial sediment, resuspended with the PBS damping fluid, the high-pressure homogeneous appearance of APV is with broken 3 times of 1500bar circulation; 4 ℃ of centrifugal 15min of 8000g collect supernatant (broken bacterium liquid supernatant), put-70 ℃ of preservations.The 50%PEG solution (50%PEG solution: 50gPEG6000 (Ameresco company) is dissolved in 100ml ddH2O) of 1 times of volume is slowly joined in the above-mentioned broken bacterium liquid supernatant of 4 times of volumes 4 ℃ of stirred overnight.Second day, 4 ℃ of 8000rpm, 30min is centrifugal, removes supernatant, collecting precipitation.With the resuspended deposition of aseptic PBS, be concentrated into 1/10 of sample original volume, obtain concentrating sample (experimental group concentrating sample, control group concentrating sample and negative control group concentrating sample).

2) purifying of recombinant C ox A16 virus-like particle: each 7ml of CsCl solution that in centrifuge tube, carefully adds 1.4g/ml, 1.32g/ml and 1.2g/ml from the bottom to top successively; Get the concentrating sample that obtains in the broken supernatant of yeast (above-mentioned steps 1)) put CsCl discontinuous density gradient (CsCl is glad through company of section available from Beijing); 4 ℃ with the centrifugal 21h of Beckman SW32Ti rotary head 100000g; The resuspended deposition of PBS damping fluid; 4 ℃ of centrifugal 15min of 13000g collect supernatant and put-70 ℃ of preservations, obtain testing sample (experimental group testing sample, control group testing sample and negative control group testing sample).

Testing sample behind the purifying is carried out protein yield through ultraviolet spectrophotometer to be measured; The result shows and to utilize the recombinant expression vector pESC-P1Y-3CDY after codon optimized provided by the present invention; In yeast expression system, every liter of fermented liquid can prepare the recombinant C ox A16 virus-like particle of about 400-500mg; And the recombinant expression vector pESC-P1W-3CDW of wild-type is in yeast expression system, and the Cox A16 virus-like particle of every liter of fermented liquid preparation is less than 10mg.The result shows the codon optimized output of Cox A16 virus-like particle in yeast expression system that effectively raises.

The evaluation and the electron microscopic observation of embodiment 4, recombinant C ox A16 virus-like particle

Testing sample (experimental group testing sample and control group testing sample) to embodiment 3 preparations carries out Western Blot evaluation and electron microscopic observation.

1, Western Blot identifies the expression of recombinant C ox A16 capsid protein: the BCA protein quantification test kit (enhancement type) that utilizes green skies company (P0010S); Operation steps to specifications, the testing sample (experimental group testing sample, control group testing sample or negative control) behind the purifying that embodiment 3 is obtained carries out protein quantification.According to quantitative result, three kinds of testing samples are respectively got an amount of suspension (the appearance Tot Prot of going up that guarantees three kinds of samples equates) carry out the 10%SDS-PAGE electrophoresis.The rabbit polyclonal antibody that uses identification Cox A16 whole protein is one anti-(one anti-stick up Divine Land Bioisystech Co., Ltd by Beijing justice provide); The fluorescent mark goat anti-rabbit antibody is that two anti-(two is anti-available from KPL, and catalog number is 072-07-15-06) carry out Western Blot analysis (albumen Marker is available from Fermentas company).

The result is as shown in Figure 5; Experimental group testing sample and control group testing sample all detect three specific bands of Cox A16 that size is about 34KD (VP1), 30KD (VP2) and 27KD (VP3) (because VP4 albumen size is merely 7KD; So be difficult to detect simultaneously), and experimental group (codon optimized back) three specific band signals of detected CoxA16 obviously are better than control group (wild-type); And the negative control group testing sample does not detect the specific band of Cox A16.

2, the electron microscopic observation of recombinant C ox A16 virus-like particle: three testing samples (experimental group testing sample, control group testing sample or negative control) adjustment protein concentration behind the purifying that embodiment 3 is obtained is consistent; Respectively get 10 μ l suspensions and drip static 1min on covering plating carbon supporting film; Blot plating carbon supporting film (available from middle mirror section appearance; Numbering: BZ110223) surperficial residual liquid, with 2% phospho-wolframic acid (available from middle mirror section appearance, numbering: the GZ02536) 1min that dyes; Blot plating carbon supporting film surface raffinate, room temperature is placed dry back with its morphological structure of transmission electron microscope observation.

The result shows; The existence of experimental group testing sample (Fig. 6) and the control group testing sample virus-like particle about all visible about 30nm of diameter under Electronic Speculum; And under the same concentrations condition, testing sample observes virus-like particle will be obviously more than control group (wild-type) testing sample for experimental group under the same Electronic Speculum visual field (codon optimized back); Negative control group (empty carrier) test sample article are not then observed the existence of virus-like particle under Electronic Speculum.

The result of present embodiment shows; The recombinant expression vector that carries Cox A16 P1 gene and Cox A16 3CD gene after codon optimized is in Yeast system; Successful packing goes out the virus-like particle of Cox A16; Compare with Cox A16 3CD gene than wild-type Cox A16 P1 gene simultaneously, improved the output of Cox A16 virus-like particle in yeast expression system after the codon optimization of the present invention.

Claims (10)

1. the preparation method of coxsackie virus A 16-type virus-like particle comprises the steps:

1) with the P1 gene of coxsackie virus A 16-type and 3CD gene clone in the purpose plasmid, obtain recombinant expression vector;

2) recombinant expression vector that obtains with step 1) transforms the purpose yeast cell, obtains expressing the recombinant yeast cell of said P1 gene and said 3CD gene;

3) cleavage step 2) recombinant yeast cell that obtains, separate the coxsackie virus A 16-type virus-like particle that obtains recombinating.

2. method according to claim 1 is characterized in that: said P1 gene and said 3CD gene are the gene that codon is optimized; Said being optimized under the prerequisite that does not change wild-type coxsackie virus A 16-type P1 albumen and wild-type coxsackie virus A 16-type 3CD Argine Monohydrochloride sequence replaces with the codon of wild-type coxsackie virus A 16-type P1 gene and wild-type coxsackie virus A 16-type 3CD gene the codon of yeast cell preference.

3. method according to claim 1 and 2 is characterized in that: the nucleotides sequence of said P1 gene is classified as in the sequence table shown in the sequence 1; The nucleotides sequence of said 3CD gene is classified as in the sequence table shown in the sequence 2.

4. method according to claim 1 is characterized in that: said purpose yeast cell is yeast saccharomyces cerevisiae, multiple-shaped nuohan inferior yeast, pichia pastoris phaff, cell wall Crewe Vickers yeast, newborn Crewe Vickers yeast or grain wine fragmentation sugar yeast.

5. method according to claim 1; It is characterized in that: each is transcribed by a promoters driven in said recombinant expression vector for said P1 gene and said 3CD gene, and the promotor direction that drives said P1 gene is in the opposite direction with the promotor that drives said 3CD gene.

6. according to arbitrary described method among the claim 1-5, it is characterized in that: each transcribes the recombinant plasmid that obtains by a promoters driven to said recombinant expression vector for two MCS places said P1 gene and said 3CD gene being inserted into pESC-URA respectively.

7. utilize the coxsackie virus A 16-type virus-like particle that arbitrary said method prepares among the claim 1-6.

8. the P1 gene of the coxsackie virus A 16-type of codon optimized type is characterized in that: the nucleotides sequence of said P1 gene is classified as in the sequence table shown in the sequence 1; And/or

The 3CD gene of the coxsackie virus A 16-type of codon optimized type is characterized in that: the nucleotides sequence of said 3CD gene is classified as in the sequence table shown in the sequence 2.

9. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain said P1 gene of claim 8 and/or 3CD gene.

Said P1 gene of claim 8 and/or 3CD gene preparation following a) or b) application in the product:

A) coxsackie virus A 16-type virus-like particle;

B) with the coxsackie virus A 16-type virus-like particle be the preventative vaccine of activeconstituents.

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