CN102465137B - Pichia pastoris for expressing rotavirus expression particles as well as preparation method and application of pichia pastoris - Google Patents
Pichia pastoris for expressing rotavirus expression particles as well as preparation method and application of pichia pastoris Download PDFInfo
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Abstract
The invention relates to pichia pastoris for expressing rotavirus expression particles as well as a preparation method and application of the pichia pastoris, and discloses a human rotavirus structural protein expression cassette, comprising the following elements: (a) an initial signal element AOX; (b) a rotavirus structural protein gene; and (c) a termination signal element TT. The invention further discloses a yeast cell transfected by using the expression cassette, a method for preparing the rotavirus viroid particles by using the yeast cell, viroid particles prepared by using the method, and application of the viroid particles for preparing vaccine compositions for preventing or treating rotavirus infection. The invention also discloses a preparation method of the yeast cell.
Description
Invention field
The present invention relates to field of genetic engineering, particularly, utilize Yeast system to express rotavirus structural protein, assembling rotavirus viruslike particle, for preventing rotavirus infection.
Background of invention
Rotavirus is one of sick modal pathogenic agent of global infant's severe diarrhea, its main infection intestinal epithelial cell, thus cause cell injury, cause diarrhoea.Rotavirus is popular in autumn and winter in summer every year, and route of infection is excrement-mouth approach, and clinical manifestation is acute gastroenteritis, is osmotic diarrhea disease, and the course of disease is generally 7 days, and heating continues 3 days, vomits 2～3 days, suffers from diarrhoea 5 days, seriously occurs dewatering symptom.
Rotavirus always has seven kinds, is numbered A, B, C, D, E, F and G etc. respectively with English alphabet.The mankind are mainly the infection that is subject to rotavirus A kind, B kind and C kind, and wherein modal be the infection of rotavirus A kind.And these seven kinds of rotaviruss all can cause disease with it other animals.
Among rotavirus A kind, there is different virus strain, be referred to as serum variant (serovar).Similar with influenza virus, rotavirus has been used dual categorizing system, according to two structural protein matter of virosomal surface, does to classify.Glycoprotein VP7 has defined G type.For protease-sensitive protein VP4, defined P type.P type can indicate P serotype with a numeral, and indicates corresponding P genotype by a numeral of square bracket inside.The method for expressing of G serotype is also very similar, but the genotypic numeral of G can be identical with the numeral of G serotype.For one thing, " Rotavirus Wa strain virus strain " (rotavirus strain Wa) will be labeled as " P1A[8] G1 ".Because these two determine that G type can separately be transmitted and produce offspring with the gene of P type, so the different combination of two genes will produce various virus strain.
The genome of rotavirus has comprised 11 unique Yeast Nucleic Acid Double-helical molecules, always has 18,555 nucleoside bases pair in these 11.Each spiral or segmentation are genes, and are 1 to 11 according to the descending number consecutively of molecular dimension.Each gene can be encoded into a kind of protein, and wherein the 9th gene and the 11st genetic comparison special, they can be encoded into two kinds of protein.Yeast Nucleic Acid periphery is to have surrounded three layers of icosahedral Protein capsid.The about diameter 76.5nm of virion, and there is no peplos.There have been six viral protein frameworks whole virion (virosome).These structural protein are called as respectively VP1, VP2, VP3, VP4, VP6 and VP7.Except these structural protein, also have six unstructuredness albumen (nonstructural protein, NSP), these six albumen are only manufactured in the cell of rotavirus infection, and do not form the structure of virosome.These six unstructuredness albumen are called NSP1, NSP2, NSP3, NSP4, NSP5 and NSP6.
In coded 12 protein of rotavirus gene group, at least 6 meetings are combined with Yeast Nucleic Acid.These albumen are not also understood at present completely at rotavirus replication time institute role; It is likely relevant with packing to the synthetic of Yeast Nucleic Acid in virosome that their function is considered to, or to messenger RNA(mRNA) to be delivered to the genosome scene of copying relevant, or translate with generegulation relevant to messenger RNA(mRNA).
Structural protein:
VP1 albumen is positioned at virosome core, is a kind of ribonucleic acid polymerase.In infected cell, this kind of enzyme can produce the synthetic required messenger RNA(mRNA) of virus protein and transcribe duplicate, and produces the virosome use that rotavirus gene body Yeast Nucleic Acid fragment copies to provide new generation.
VP2 albumen forms the core layer of virosome, and bind rna genosome.
VP3 albumen is a part for virosome kernel, and is a kind of enzyme that is called guanylate transferase (guanylyl transferase).This kind of enzyme is a kind of capping enzyme (Capping enzyme), the enzyme of 5 ' end cap used when namely making messenger RNA(mRNA) post transcriptional modificaiton.This 5 ' end cap protects viral messenger RNA(mRNA) to avoid nuclease (lytic enzyme that the nucleic acid of take is substrate) hydrolysis, enables to keep stable.
VP4 albumen is positioned at the surface of virosome, outstanding and become a furcella (spike).It is combined with cell surface receptor, relevant with viral adherent cell.Before virus is infectious, VP4 albumen can be changed over VP5* albumen and VP8* albumen by a kind of proteolytic enzyme that can find at internal organ.VP4 albumen has determined this viral toxicity (Virulence), and it has also determined the P serotype that this is viral.
VP6 albumen forms housing.It is the protein that antigenicity is strong, and can be used to differentiate the kind of rotavirus.This protein is undertaken in the detection test of rotavirus A kind infection by laboratory.
VP7 albumen is a kind of glycoprotein that forms the outer capsid of virosome.Except the function on virus structure, it also determines the G serotype of this virus strain.VP7 protein is the same with VP4 protein, is all the main target of immunity, is the main path of protecting from infection.
Rotavirus strain has very big-difference in various places, but the mankind's rotavirus disease mainly causes by 5 kinds of serotypes, and wherein G1P8 and G3P8 serotype be take as main in China.Now the Rotavirus Vaccine of listing mainly be take people-animal reassortant and animal attenuated strain living vaccine as main, and this class vaccine application can cause the generation of the serious side reactions such as intussusception.The infection being produced by rotavirus different serotypes, the intersecting protective of vaccine may be a little less than, and the issuable serious side reaction of vaccine at present, the needs that strongly need new Rotavirus Vaccine to meet treatment and prevent.
Invention summary
Therefore, an object of the present invention is to provide a kind of human rotavirus's viruslike particle of new serotype as the candidate vaccine molecule of rotavirus infection.
In one aspect of the invention, disclose a kind of human rotavirus's structural protein expression cassette, comprised following element:
(a) start signal element AOX;
(b) rotavirus structural protein gene;
(c) termination signal elements T T.
Preferred described rotavirus vp 2 is encoded by SEQ ID NO:1; Described rotavirus vp 4 is encoded by SEQ ID NO:5; Described rotavirus vp 6 is encoded by SEQ ID NO:2; Or described rotavirus VP 7 is encoded by SEQID NO:3 or SEQ ID NO:4.
Aspect second of the present invention, the yeast cell of the above-mentioned expression cassette transfection of a kind of use is provided, it is characterized in that, in the karyomit(e) of described yeast cell, be integrated with above-mentioned expression cassette, described expression cassette is expressed rotavirus vp 2, VP4, VP6 and VP7 structural protein, described yeast cell is expressed rotavirus structural protein under methanol induction, and the being made by manufacturers or users in described yeast of described rotavirus structural protein becomes viruslike particle.
In a preference aspect this, yeast cell is selected from: pichia spp, yeast saccharomyces cerevisiae or debaryomyces hansenii.Preferred, yeast cell is selected from Pichia pastoris GS115 bacterial strain and KM71 bacterial strain.
In another preference aspect this, expression cassette inserts pPIC3.5K plasmid.
The 3rd aspect of the present invention provides a kind of method of preparing rotavirus viruslike particle, comprises step:
(i) under conditions suitable for the expression, cultivate above-mentioned yeast cell, thereby express rotavirus structural protein VP2, VP4, VP6 and VP7; With
(ii) from culture, isolate viruslike particle.
The 4th aspect of the present invention provides the viruslike particle of preparing with aforesaid method, and it is comprised of rotavirus vp 2, VP4, VP6 and VP7 albumen.
In a preference aspect this, VP7 albumen is selected from G1 or G3 serotype.
In another preference aspect this, VP4 albumen is P8 serotype.
In another preference aspect this, rotavirus vp 2 is SEQ ID NO:8; Described rotavirus vp 4 is SEQ ID NO:12; Described rotavirus vp 6 is SEQ ID NO:9; Or described rotavirus VP 7 is selected from the aminoacid sequence of SEQ ID NO:10 and SEQ ID NO:11.
The 5th aspect of the present invention provides the purposes of viruslike particle, for the preparation of the vaccine composition of prevention or treatment rotavirus infection.
The 6th aspect of the present invention, provides the preparation method of above-mentioned yeast cell, comprises step:
A) with above-mentioned expression cassette, be inserted in pPIC3.5K plasmid, make plasmid contain the expression cassette of rotavirus vp 2, VP4, VP6 and VP7 structure gene simultaneously;
B) with transformed yeast cell together with the plasmid of described expression rotavirus vp 2, VP4, VP6 and VP7, be integrated in yeast chromosomal, thereby obtain the yeast cell of above-mentioned production rotavirus viruslike particle.
Accompanying drawing summary
Fig. 1 has shown the western blot analysis of the broken bacterium liquid of restructuring G1P8 yeast.Wherein each swimming lane is 1: negative yeast contrast; 2: albumen Marker; No. 3-6:1-No. 44 saccharomycetic broken bacterium liquid of restructuring G1P8.
Fig. 2 has shown the sucrose density gradient centrifugation analysis of recombination microzyme.Wherein each swimming lane is as follows: 1: negative control; 2: protein labeling (Marker); 3-7: negative yeast gradient centrifugation is received sample; 8: protein labeling; The centrifugal loading former state of 9:1 yeast; 10-14:1 yeast gradient centrifugation is received sample; The centrifugal loading former state of 15:2 yeast; 16-20:2 yeast density gradient centrifugation is received sample; The centrifugal loading former state of 21:3 yeast; 22-24,25: protein labeling; 26-27:3 yeast density gradient centrifugation is received sample; The centrifugal loading former state of 28:4 yeast; 29-33:4 yeast density gradient centrifugation is received sample.Wherein the possibility of No. 1 and No. 4 yeast clone formation VLP particle is larger.
Fig. 3 has shown restructuring G1P8 yeast (GS115 bacterial strain, No. 1 clone's) molecular sieve analysis.
Fig. 4 has shown the western blotting evaluation that restructuring G1P8 yeast (No. 1 clone) molecular sieve is received sample.Each swimming lane sample is as follows: 1:G1P8 yeast breaks bacterium supernatant; 2: broken bacterium supernatant ultrafiltration and concentration; 3: liquid is abandoned in ultrafiltration; 4: protein labeling; 5-8: first protein peak of molecular sieve; 9-19: second peak of molecular sieve; 13: protein labeling.The sample that the 1st of molecular sieve and the 2nd protein peak are collected is identified through western blotting, and the visible rotavirus vp 6 protein-specific bands (41000) of receipts sample of the 1st protein peak may comprise the VLP of formation; The receipts sample of the 2nd protein peak has no specificity rotavirus vp 6 protein bands.
Fig. 5 has shown the molecular sieve analysis of restructuring G1P8 yeast (KM71 bacterial strain) expressing protein.
Fig. 6 has shown the western blotting evaluation of restructuring G1P8 yeast (KM71 bacterial strain) molecular sieve receipts samples.Wherein each swimming lane sample is as follows: 1:G1P8 yeast breaks bacterium liquid ultrafiltration and concentration (loading after dilution in 1: 5); Before the broken bacterium liquid ultrafiltration of 2-:G1P8 yeast; 3: protein labeling; 4: protein labeling; 5-13: first protein peak of molecular sieve is received sample; 14: negative control; 15: protein labeling; 16-23: second protein peak of molecular sieve received sample.Broken bacterium liquid is through western blot analysis, and visible molecular sieve the 1st and the 2nd peak all contain rotavirus vp 6 albumen.
Fig. 7 a has shown in Fig. 3 that first protein peak and Fig. 7 b have shown the transmission electron microscope observing of first protein peak (* 30000) result in Fig. 5.In first protein peak sample, all visible diameter between 50-100nm, as arrow indication, big or small inhomogenous virus-like particle structure.In the 2nd protein peak of Fig. 3 and Fig. 5, be showed no obvious grain pattern.
Embodiment
1. viruslike particle
The viruslike particle obtaining by outer-gene recombinant technology (Virus-like particle, VLP) have natural viral external structure and in and antigenic component, but do not comprise viral DNA/RNA, therefore VLP can not copy in vivo, improved the security of vaccine application, meanwhile, VLP has fabulous immunogenicity.The subunit vaccine being comprised of viruslike particle becomes the important candidate vaccine molecule of prophylaxis of viral infections gradually.Hepatitis B virus and human cytomegalic inclusion disease virus VLP are by the license of U.S. Bureau of Drugs Supervision now.
Contriver is through Nucleotide and amino acid identity comparison, choose with international standard strain and the larger place of china human rotavirus strain of Asia strain homology as VP2, VP6, VP4 (P8 serotype), the DNA profiling chain (table 1 sees below) of VP7 (G1 and G3 serotype), DNA sequence dna is adjusted according to pichia spp codon usage frequency table (table 2 sees below), built and contained human rotavirus VP2, VP4, the recombinant yeast pichia pastoris bacterium of VP6 and VP7 albumen, form the G1P8 serotype different with G3P8, and self assembly becomes viruslike particle (VLP) in Yeast system, these VLP are studied, as the instrument of further preparing vaccine.
1. development process:
The flow process of the rotavirus VLP of exploitation particular serotype is provided below:
Determine → the gene optimization of rotavirus vp 2, VP4, VP6 and VP7 protein D NA template sequence, synthesize → upstream of full gene builds plasmid, electricity is transformed into Pichi strain, forms double-deck or three layers of rotavirus particle → abduction delivering, and the downstream purification of evaluation → VLP
2. the preparation of antibody:
After having obtained the viruslike particle of complete particular serotype, by purifying, reclaim the individual protective immunity that obtains of its inoculation for these viruslike particles.The research of this immunization method and checking immunizing power can be referring to following document.[1] Offit PA, the protective immune response for rotavirus infection that the non-infectious rotavirus of Dudzik KI. (RRV strain) is induced in Mice Body (Noninfectious rotavirus (strain RRV) induces an immune response in mice which protects against rotavirus challgenge.) clinical microbiology magazine, 1989,27 (5): 885-888; [2] McNeal MM, Sheridan JF, the immunity of Ward RL. intraperitoneal is for provide protection (the Active protection against rotavirus infection of mice following intraperitoneal immunization.) Journal of Virology of mouse rotavirus infection, 1992,191 (1): 150-157; [3] Ciarlet M, Crawford SE, Barone C, Deng. the protective immunity that rotavirus subunit vaccine is induced through parenteral immunizing rabbit (Subunit rotavirus vaccine administered parenterally to rabbits induces active protective immunity.) Journal of Virology, 1998,72 (11): 9233-9246; [4] O ' Neal CM; Crawford SE; Estes MK; Deng; rotavirus sample particle is through protective response (the Rotavirus virus-like particles administered mucosally induce protective immunity.) Journal of Virology of mucosal immunity induction; 1997,71 (11): 8707-8717.
3. composition
Viruslike particle of the present invention can become composition with other component co-formulated.For example, viruslike particle of the present invention and adjuvant are mixed together, or mix before using, or can before or after adjuvant, use, to improve immunizing power.Viruslike particle can also mix with other added ingredients, such as acceptable carrier, liposome, stabilizing component, pigment, seasonings etc. on solvent, antiseptic-germicide, pharmacology, is mixed together, and to facilitate, uses.
4. medicine box
Viruslike particle of the present invention, and composition can also be made medicine box for being applied to the immune individuality of needs.Medicine box can contain specification sheets, to indicate application program.Each component in medicine box can be placed in different vessels, also can mix.These compositions can be simultaneously, successively or interval use.Utilize viruslike particle immune body, and various immunization protocol is all confirmable in those skilled in the art's horizontal extent.
5. Equivalent
Those skilled in the art answer, and maybe can guarantee to be no more than use normal experiment, understand many equivalents of invention embodiment as herein described.
All publications, patent and the patent application of mentioning in this specification sheets all introduced with identical degree in specification sheets, and as each independent publication, patent or patent application, introducing is the same in this article especially with separately.
Embodiment
Materials and methods:
1 plasmid and bacterial strain
PPIC3.5K plasmid (purchased from Invitrogen), intestinal bacteria top10, Pichia pastoris GS115 bacterial strain and pichia spp KM71 bacterial strain are purchased from Invitrogen company.
2. reagent
T4 DNA ligase, restriction enzyme EcoR I, Not I, Mlu I, Bgl II, BspE I and BamH I are purchased from Fermentas company, DNA marker and protein labeling are Fermentas company product, DNA gel reclaims test kit and plasmid extraction test kit is Axygen company product, anti-VP6 monoclonal antibody-rotavirus capsid (2B4) is Santa Cruz product, and DEAE Sepharose FF chromatographic stuffing and AKTA purification system are purchased from GE company.
The optimization of embodiment 1 rotavirus vp protein structure gene and synthetic
By Blast software analysis, through Nucleotide and amino acid identity comparison, choose and international standard strain and the larger place of china human rotavirus strain of the Asia strain homology DNA profiling chain (table 1) as VP2, VP6, VP4 (P8 serotype), VP7 (G1 and G3 serotype).DNA sequence dna is according to pichia spp codon usage frequency table (table 2), the whole synonyms of goal gene replaced, and removed some AT and be rich in district, and adjusting GC content is 45-50%.Restriction enzyme site relevant with construction of recombinant plasmid in sequence (EcoR I, Not I, Mlu I, Bgl II, BspE I and BamH I) is carried out to same sense mutation simultaneously.5 ' end adds Kozak (ACCATG or ACCGCCATG) sequence, and 3 ' end adds terminator codon.Send Jin Site bio tech ltd, Nanjing synthetic the structure gene of each VP albumen of having optimized.
Choosing of table 1 human rotavirus VP structural protein template strand
Structural protein template
VP2/VP6 albumen people Wa strain
VP7 albumen (G1 serotype) Chi-87 strain
VP7 albumen (G3 serotype) Beijing 97 ' S48 strain
VP4 albumen (P8 serotype) 97 ' B53 strain
Table 2 pichia spp preference codon uses table
| Albumen | Optimal codon | Albumen | Optimal codon |
| Gly(G) | GGT | Lys(K) | AAG |
| Glu(E) | GAG | Asn(N) | AAC |
| Asp(D) | GAC | Met(M) | ATG |
| Val(V) | GTC | Ile(I) | ATC |
| Ala(A) | GCT | Thr(T) | ACT |
| Arg(R) | AGA | Trp(W) | TGG |
| Ser(S) | TCC | Cys(C) | TGT |
| Tyr(Y) | TAC | Gln(Q) | CAG |
| Leu(L) | TTG | His(H) | CAC |
| Phe(F) | TTC | Pro(P) | CCA |
The gene coded sequence of each VP structure gene and aminoacid sequence are as described in sequence table.Wherein rotavirus vp 2 is encoded by SEQ ID NO:1; Rotavirus vp 4 is encoded by SEQ ID NO:5; Rotavirus vp 6 is encoded by SEQ ID NO:2; Rotavirus VP 7 is by SEQ ID NO:3 (G1) or SEQ ID NO:4 (G3) coding.Its aminoacid sequence is respectively: rotavirus vp 2 is SEQ ID NO:8; Rotavirus vp 4 is SEQ ID NO:12; Rotavirus vp 6 is SEQ ID NO:9; Rotavirus VP 7 is selected from SEQ ID NO:10 (G1) and SEQ ID NO:11 (G3).
The transformation of embodiment 2pPIC3.5K expression plasmid
By the pPIC3.5K obtaining in VP2, the VP6, VP4 (P8 serotype), VP7 (G1 or G3 serotype) gene fragment and the embodiment 2 that obtain in above-described embodiment 1
changeplasmid all carries out double digestion by restriction enzyme EcoR I and Not I, reclaims test kit reclaim 5 VP gene fragments and pPIC3.5K with DNA gel
changethe enzyme of carrier is cut product.According to plasmid and object fragment mol ratio 1: 3, with 22 ℃ of connections of T4DNA ligase enzyme, spend the night, connecting product be 5 recombinant plasmids with 5 ' AOX-VP gene-TT expressed intact box, distinguishes called after pPIC3.5K
change-VP2, pPIC3.5K
change-VP6, pPIC3.5K
change-VP4 (P8 type), pPIC3.5K
change-VP7 (G1 type), pPIC3.5K
change-VP7 (G3 type).Recombinant plasmid transforms respectively intestinal bacteria top10 competent cell, is then applied on the LB agar plate containing penbritin (500 μ g/ml) 37 ℃ of overnight incubation.Second day picking mono-clonal bacterium, and serve the order-checking of Hai Shenggong biotechnology company limited.
The structure of embodiment 4 restructuring G1P8 plasmids and G3P8 plasmid
By the pPIC3.5K obtaining in embodiment 3
change-VP2 plasmid carries out double digestion, pPIC3.5K by Bgl II and Mlu I
change-VP6 plasmid carries out double digestion by BamH I and Mlu I, reclaims respectively enzyme cut product with DNA gel recovery test kit, then connects conversion reaction, and coating is containing the LB agar plate of penbritin.Picking mono-clonal bacterium, with test kit extracting plasmid, identifies through Bgl II and Mlu I double digestion, builds and obtains correct pPIC3.5K
change-VP6-VP2 plasmid.PPIC3.5K
change-VP6-VP2 plasmid carries out double digestion, pPIC3.5K by Bgl II and Mlu I
change-VP7 (G1 type) plasmid carries out double digestion by BamH I and Mlu I, through connecting to transform to build, obtains pPIC3.5K
change-VP7-VP6-VP2 plasmid.PPIC3.5K
change-VP4 (P8 type) plasmid carries out double digestion, pPIC3.5K by Bgl II and Mlu I
change-VP7-VP6-VP2 plasmid carries out double digestion by BamH I and Mlu I, through connecting to transform to build, obtains pPIC3.5K
change-VP7-VP6-VP2-VP4 plasmid, the G1P8 plasmid of recombinating.In like manner, build the G3P8 plasmid that obtains recombinating.
Abduction delivering and the evaluation of embodiment 5 recombination microzyme G1P8
By the restructuring G1P8 plasmid obtaining in embodiment 4 and empty plasmid pPIC3.5K after the linearizing of BspE I single endonuclease digestion, through 1500V/5ms electric shock, proceed in Pichia pastoris GS115 bacterial strain and KM71 bacterial strain competent cell, be laid on MD flat board, cultivate after 3-5 days, grow His for 28 ℃
+transformant.4 clones of picking from transformant, are inoculated in 5ml BMGY substratum (1.34%YNB, 0.00004% vitamin H, 1% glycerine, 100mM potassium phosphate buffer, PH 6 for 2% peptone, 1% yeast extract), and 28 ℃, 250r/min, overnight incubation.Second day, treats A
600while reaching 2-6,1500r/min, centrifugal 5min, quantitatively draws yeast cell, and 10ml BMMY substratum is resuspended, adjusts A
600be 1.28 ℃, 250r/min, supplement 0.5% methyl alcohol every day, mends continuously three days, and within the 4th day, the centrifugal 3min of 12000r/min collects thalline ,-80 ℃ of preservations.Broken damping fluid (the 100mM NaH of 500 μ l
2pO
42H
2o, 1mM EDTA, 5% glycerine, PH7.4) resuspended thalline, adds the broken bacterium of stainless shot (Ao Ran bio tech ltd, Shanghai) machinery, 12000r/min, centrifugal 10min, collects supernatant.Broken bacterium liquid and 4 * SDS sample-loading buffer mix, 98 ℃ of water-bath 10min.Loading is carried out 12%SDS-PAGE protein electrophoresis, running gel 100V, and 1h transfers on pvdf membrane, and 5% skimmed milk sealing is spent the night, and adds the monoclonal antibody (1: 450) of the anti-VP6 of mouse in incubated at room 1.5h.With PBS-0.05%Tween20 (traditional Chinese medicines chemical reagent company limited), wash after film three times, with 26 ℃ of effect 1h of the anti-mouse IgG of rabbit (1: 5000) of HRP mark, the same washing after film three times, with the colour developing of HRP Color Appearance System.
In centrifuge tube, spread respectively the broken bacterium supernatant liquor of 2ml 58% sucrose, 2ml30% sucrose and 2ml, 32000r/min, 4 ℃, ultracentrifugation 4h.At the bottom of centrifuge tube, Zha Dong receives sample, every pipe 1ml, and each sample is received sample 5 pipes altogether.All samples is the same carries out western blotting evaluation.
As mentioned above, picking 4 restructuring GS115 yeast clones, broken bacterium liquid is identified through western blotting with the monoclonal antibody of the anti-VP6 of mouse, at relative molecular weight, is the 41 000 visible specific bands in place, obtains 3 positive colonies, sees Fig. 1.Sucrose density gradient centrifugation is analyzed above-mentioned 4 clones, and the possibility of No. 1 and No. 4 yeast clone formation VLP particle is larger, sees Fig. 2.So No. 1 clone of picking carries out follow-up fermentor tank abduction delivering and molecular sieve purification analysis.
The abduction delivering of embodiment 6 restructuring yeast strains and molecular sieve preliminary purification
Restructuring GS115 yeast engineering strain of picking, carries out scale fermentation.600ml Breaking buffer (100mM NaH for fermentation thalline (about 120g)
2pO
42H
2o, 2mM MgCl
2, 5% glycerine, PH7.4) resuspended, with the broken bacterium of high-pressure cell crusher (Constant System Limited), 30Kpsi, broken bacterium 2 times.In broken bacterium liquid, add 500 μ g/ml DNA enzymes, 1mmol/L PMSF and 0.16%Triton-X-100 (traditional Chinese medicines chemical reagent company limited), 4 ℃, mild stirring is spent the night again.10000g, 20min, 4 ℃ are centrifugal, get supernatant liquor, with 0.45 μ m filter membrane micro-filtration, remove cell debris, then ultrafiltration and concentration, and sample is again through the standby sieve chromatography purity analysis of doing of 0.45 μ m membrane filtration.The results are shown in Figure 3.
Level pad for post (PBS, 400mMNaCl, 0.02%Tween-80) balance, the direct upper prop of sample, collects effluent liquid.The Sepharose 4FF chromatography column that we adopt can be separated molecular size scope at 60000-20000000, particle diameter is at 45-165 μ m, and rotavirus VLP diameter is 75nm, relative molecular weight is 7.2 * 10
7da, is therefore applicable to carrying out sieve chromatography.And the VLP forming after wash-out is due to first exclusion outflow of particle diameter conference.The sample that the 1st of molecular sieve and the 2nd protein peak are collected identifies through western blotting, and the first exclusion that after sieve chromatography, molecular weight is large goes out peak, and the rear peak that washed out that molecular weight is little, is shown in Fig. 3.Adopt the specific antibody of VP6 to identify, the visible specific band of receipts sample (41000) at the 1st exclusion peak, therefore may comprise the VLP that assembling forms, the receipts sample of the 2nd elution peak is in Fig. 49 Dao He 10 roads, have no specific band, can be the VP albumen of micromolecular unassembled one-tenth particle or pichia spp host's albumen.
In like manner, proceed to KM71 Host Strains, carry out abduction delivering and molecular sieve purification analysis, two protein peaks the results are shown in Figure 5.Broken bacterium liquid is through western blot analysis, and all there are rotavirus vp 6 specific proteinses at visible molecular sieve the 1st and the 2nd peak.The same, the visible specific band of receipts sample (41000) at the 1st exclusion peak, may comprise the VLP that assembling forms, and specific band is also shown in by the receipts sample of the 2nd elution peak, but may be for having neither part nor lot in the VP albumen of assembling or pichia spp host's albumen.
The transmission electron microscope observing of embodiment 7 recombination microzyme G1P8 VLP
The protein peak sample that sieve chromatography purifying is collected is placed in 60s on the coated copper mesh of polyvinyl formal carbon, and with 1% acetic acid uranium dyeing 30s, under room temperature, (22 ℃) dry 10min, adopts the formation of transmission electron microscope observing VLP particle.
The 1st protein peak in molecular sieve analysis chart 3 and the 1st protein peak sample in Fig. 5, through the visible diameter of transmission electron microscope observing between 50-100nm, as arrow indication, big or small inhomogenous virus-like particle structure (Fig. 7 a and Fig. 7 b), has no obvious grain pattern in the 2nd protein peak.
But above-mentioned the specific embodiment of the present invention is only used to describe, it will be understood by those skilled in the art that and can carry out many changes to details, and without prejudice to the invention described in claims.
All publications, patent and the patent application of mentioning in this specification sheets all introduced with identical degree in specification sheets, and as each independent publication, patent or patent application, introducing is the same in this article especially with separately.
Claims (4)
1. the yeast cell of a transfection, described yeast cell is pichia spp, it is characterized in that, in the karyomit(e) of described yeast cell, be integrated with expression cassette, described expression cassette is expressed rotavirus vp 2, VP4, VP6 and VP7 structural protein, described yeast cell is expressed rotavirus structural protein under methanol induction, and the being made by manufacturers or users in described yeast of described rotavirus structural protein becomes viruslike particle, wherein:
Described expression cassette is human rotavirus's structural protein expression cassettes, comprises following element:
(a) start signal element AOX;
(b) gene of coding colyliform virus structural protein VP2, VP4, VP6 or VP7;
(c) termination signal elements T T, wherein said rotavirus structural protein VP2 is encoded by SEQ ID NO:1; Described rotavirus structural protein VP4 is encoded by SEQ ID NO:5; Described rotavirus structural protein VP6 is encoded by SEQ ID NO:2; Or described rotavirus structural protein VP7 encodes by SEQ ID NO:3 or SEQ ID NO:4, and
Described expression cassette inserts pPIC3.5K plasmid.
2. yeast cell as claimed in claim 1, is characterized in that, described yeast cell is selected from Pichia pastoris GS115 bacterial strain and KM71 bacterial strain.
3. a method of preparing rotavirus viruslike particle, is characterized in that, described method comprises step:
(i) under conditions suitable for the expression, cultivate yeast cell claimed in claim 1, thereby express rotavirus structural protein VP2, VP4, VP6 and VP7; With
(ii) from culture, isolate viruslike particle.
4. the preparation method of yeast cell claimed in claim 1, is characterized in that, described method comprises step:
A) with human rotavirus's structural protein expression cassette, be inserted in pPIC3.5K plasmid, make plasmid contain the expression cassette of rotavirus vp 2, VP4, VP6 and VP7 structure gene simultaneously, described expression cassette comprises following element:
(a) start signal element AOX;
(b) gene of coding colyliform virus structural protein VP2, VP4, VP6 or VP7;
(c) termination signal elements T T, wherein said rotavirus structural protein VP2 is encoded by SEQ ID NO:1; Described rotavirus structural protein VP4 is encoded by SEQ ID NO:5; Described rotavirus structural protein VP6 is encoded by SEQ ID NO:2; Or described rotavirus structural protein VP7 is encoded by SEQ ID NO:3 or SEQ ID NO:4;
B) with transformed yeast cell together with the plasmid of described expression rotavirus structural protein VP2, VP4, VP6 and VP7, be integrated in yeast chromosomal, thereby obtain the yeast cell of production rotavirus viruslike particle claimed in claim 1.
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