CN105247037A

CN105247037A - Porcine circovirus (pcv2) subunit vaccine using recombinant yeast whole cells and method for manufacturing same

Info

Publication number: CN105247037A
Application number: CN201480026972.6A
Authority: CN
Inventors: 崔毅星; 朴景民; 徐成和; 安廷梧; 李殷教; 金千锡; 尹寅重; 俞成植; 沈宁贞
Original assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB; CHOONG ANG VACCINE LAB
Current assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB; CHOONG ANG VACCINE LAB
Priority date: 2013-03-11
Filing date: 2014-03-11
Publication date: 2016-01-13
Also published as: WO2014142515A1; KR20140111541A; KR102217387B1

Abstract

The present invention relates to a recombinant porcine circovirus (PCV2) subunit vaccine using yeast whole cells or lysates thereof and a method for manufacturing the same. The yeast whole cells or lysates thereof according to the present invention have an excellent effect as a vaccine composition, and can also significantly simplify processes, such as cell disruption, antigen extraction, purification, stabilization, and the like, which cannot be avoided in a procedure for manufacturing a porcine circovirus vaccine using various advantages of yeast and recombinant microorganisms.

Description

Use pig circular ring virus (PCV2) subunit vaccine and the manufacture method thereof of the full cell of recombination yeast

Technical field

The present invention relates to for being pig circular ring virus (PCV2) subunit vaccine of sow and the vaccinated restructuring of piglet and production method thereof, pig circular ring virus (PCV2) subunit vaccine of this restructuring comprises the whole yeast cells of restructuring and can prevent the disease that pig circular ring virus (PCV) is relevant.

Background technology

2 porcine circovirus (PCV-2) is known is the virus in the principal causative source of postweaning multisystem exhaustion syndrome (PMWS).The reason being difficult to develop the vaccine that pig circular ring virus can be prevented to damage is, use zooblast to produce vaccine and need relatively costly substratum and animal cell culture systems, and contained various heterologous proteins likely during inoculation have side effects in blood serum medium.

Four kinds of PCV2 vaccines based on the ORF2 antigen from PCV2a virus are just sold on world market.In foreign country, the company being in highest level is technically Cimmeria (Merial, France), and there be a lot of patent relevant to PCV in the said firm, and uses the PCV2 totivirus (Circovac) through deactivation.Boehringer Ingelheim (BoehringerIngelheim) develops a kind of by expressing the vaccine based on recombinant protein (avoiding PCV and Ciroflex) that ORF2 obtains in baculovirus, and wherein the advantage of this vaccine is injected with the amount of 1ml after being purifying at every turn.Intervet (Intervet) company limited passes through to express ORF2 in baculovirus and purified expression product also develops a kind of vaccine based on recombinant protein, wherein this vaccine is designed to the interval of 3 weeks, to be expelled in piglet body twice with the amount of 2ml.FortDodge company limited develops a kind of mosaic vaccine by the ORF2 expressing PCV2 in PCV-1 virus, wherein this mosaic vaccine with an amount shot of 2ml in piglet body.

When the subunit vaccine of recombinant virus, the antigenicity that formation virus-like particle (VLP) can increase vaccine is known.When yeast, develop the technology utilizing the Ty1 in yeast saccharomyces cerevisiae (S.cerevisiae) bacterial strain (a kind of for a long time just known before retrotransposition sub-element).Ty1 on gene, structure and function with retroviral nucleosides/nuclear phase seemingly.The vaccine using Ty1 to obtain is reported in 1987, and when Vaccine candidate gene and Ty1 gene 3 ' area merges and when expressing, have the following advantages: do not hinder the formation of virus-like particle and virus-like particle can be formed in cell, and not needing the factor that other is separated.Several sections of reports are had to point out: when producing vaccine in yeast, yeast cell wall fraction also can be used as adjuvant (ChanGC, ChanWK, SzeDM.2009, Theeffectsofbeta-glucanonhumanimmuneandcancercells.JHema tolOncol2:25).

If it is known that cracking yeast cell is to be extracted in the antigen of cells, then after preparing cell pyrolysis liquid, the antigen of such as virus-like particle is difficult to stably keep in the solution.In addition, also may need various additive (such as, the tensio-active agent of such as polysorbate20, the such as sugar of sucrose, trehalose or Sorbitol Powder, etc.), prevent virus-like particle from occurring to assemble and precipitation (LangR, WinterG, VogtL, ZuercherA, DorigoB, SchimmeleB (2009)).The recombiant vaccine being developed the use whole yeast cells for overcoming above-mentioned shortcoming can be enjoyed yeast and can express one or more antigens, have high stability and be easy to carry out mass-produced advantage.

The present inventor has been found that the whole yeast cells vaccine of expressing pig circular ring virus antigen ORF2 has very high immunogenicity, thus completes the present invention.

Summary of the invention

Technical problem

Target of the present invention is to provide a kind of whole yeast cells of expressing recombinant vectors, or the lysate of this cell, comprise the pig circular ring virus vaccine composition of cell or its lysate, and their production method, wherein recombinant vectors comprises the gene ORF2 of the II type capsid protein of coding pig circular ring virus.

Technical scheme

In an embodiment of the invention, provide a kind of whole yeast cells for pig circular ring virus vaccine, described whole yeast cells comprises the II type capsid protein ORF2 of pig circular ring virus.In this embodiment, gene ORF2 can be the gene represented by SEQIDNO:1.Yeast is used as host cell, and can be the bacterial strain of yeast saccharomyces cerevisiae (Saccharomycescerevisiae).Particularly, described yeast can be Y2805 bacterial strain.In addition, described whole yeast cells can through hot deactivation or through Formalin inactivation.

In another embodiment of the present invention, provide a kind of lysate of the whole yeast cells for pig circular ring virus vaccine, the lysate of described whole yeast cells comprises the II type capsid protein ORF2 of pig circular ring virus.Gene ORF2 can be the gene represented by SEQIDNO:1.Yeast is used as host cell, and can be the bacterial strain of Saccharomyces Cerevisiae in S accharomycescerevisiae.Particularly, described yeast can be Y2805 bacterial strain.In addition, described whole yeast cells can through hot deactivation or through Formalin inactivation, and the lysate of described whole yeast cells can derive from through hot deactivation or the whole yeast cells through Formalin inactivation.

In yet, provide a kind of pig circular ring virus vaccine composition, described pig circular ring virus vaccine composition comprises the lysate of whole yeast cells or this cell, and this whole yeast cells comprises the II type capsid protein ORF2 of pig circular ring virus.In this embodiment, gene ORF2 can be the gene represented by SEQIDNO:1, and ORF2 albumen can be the polypeptide of the aminoacid sequence comprising SEQIDNO:2.Further, yeast is used as host cell, and can be the bacterial strain of Saccharomyces Cerevisiae in S accharomycescerevisiae.Particularly, described yeast can be Y2805 bacterial strain, and can lack gal operon (gal) 80 gene.In addition, described whole yeast cells can through hot deactivation or through Formalin inactivation.Can comprise concentration according to vaccine composition of the present invention is 4x10 ⁹whole yeast cells or less cell.

In yet, provide the whole yeast cells through transform of a kind of production for pig circular ring virus vaccine or the method for its lysate, described method comprises the steps: to prepare the recombinant expression vector comprising gene ORF2, the II type capsid protein of this gene ORF2 coding pig circular ring virus; With, use described recombinant vectors transformed yeast.In this embodiment, gene ORF2 can be the gene represented by SEQIDNO:1.Yeast is used as host cell, and can be the bacterial strain of Saccharomyces Cerevisiae in S accharomycescerevisiae.Particularly, described yeast can be the Y2805 bacterial strain lacking gal80 gene.In this embodiment, recombinant expression vector can comprise GAL10 promotor, and therefore ORF2 can merge with Ty1, can increase its expression.In addition, described whole yeast cells can further through hot deactivation or through Formalin inactivation.

In yet, provide a kind of for being the vaccinated method of pig, described method comprises: give pig circular ring virus vaccine composition to pig, described pig circular ring virus vaccine composition comprises the lysate of whole yeast cells or this cell, and this whole yeast cells comprises the II type capsid protein ORF2 of pig circular ring virus.In this embodiment, the genes encoding that described ORF2 albumen can be represented by SEQIDNO:1, and can be the polypeptide comprising the aminoacid sequence that SEQIDNO:2 represents.Yeast is used as host cell, and can be the bacterial strain of Saccharomyces Cerevisiae in S accharomycescerevisiae.Particularly, described yeast can be Y2805 bacterial strain, and can lack gal80 gene.In this embodiment, recombinant expression vector can comprise GAL10 promotor, and therefore ORF2 can merge with Ty1, to increase its expression.

Deactivation can be carried out according to technology well known by persons skilled in the art according to vaccine of the present invention.Described deactivation is carried out preferably by chemistry route, such as, by making antigen-exposed in the chemical reagent of such as formaldehyde (formalin), paraformaldehyde, beta-propiolactone or ethylene imine or derivatives thereof, but is not limited to this.

Term as used herein " transformant " refers to, by being incorporated in cell by foreign DNA (such as plasmid or hybrid DNA), then make this DNA in cell, carry out the cell copying and express and obtain.To the Plastid transformation of foreign DNA be comprised in yeast according to the method (NucleicAcidsResearch19,5791 (1991)) of Lithium Acetate.

Term as used herein " clone " refers to a series of process, and wherein, gene is inserted into by round pcr in the delivery system comprising plasmid or from this delivery system and shifts out, for forming new restriction site in gene.

Term as used herein " is ... vaccination " refer to following process: be injected in vivo to produce immunne response by the activeconstituents of such as albumen or antigen, thus generation memory B cell, then the tachysynthesis for same antigen is induced to reply, so that activeconstituents can be used as the treatment reagent resisting specified disease.

Term as used herein " lysis " refers to that destruction cytolemma or cell walls are with the process of release cells content (such as stirring the process of pearl and cell), and term " cell pyrolysis liquid " refers to the mixture of intracellular members and the extracellular component obtained by lysing cell.

The swine disease that the wasting syndrome that term as used herein " postweaning multisystem exhaustion syndrome (PMWS) " refers to wean in piglet is feature, this reports in multiple country (comprising Canada, the U.S., Northern Ireland, Europe, Korea S, Japan, Taiwan etc.).Postweaning multisystem exhaustion syndrome (PMWS) causes death, atrophy or body weight gains to reduce in wean piglet, and it continues the time period of 6 months to 1 year usually, thus brings serious financial loss.In Korea S, within 1997, report the first time outburst of PMWS, and PMWS caused the death of about 6,000,000 piglet in 2007, this corresponds to loss about 2,000,000,000 circle (Korea S's currency) (rural economy institute of Korea S (KoreaRuralEconomicInstitute)).

" yeast " that use in the present invention is known GRAS (the being generally considered safe) bacterial strain used safely in human body.When using yeast expression system to produce vaccine, eliminating the needs using blood serum medium, microorganism fermentation tank can be used to replace expensive animal cell culture systems, and the protein expression level in yeast cell is higher than the protein expression level in zooblast.When using yeast instead of zooblast to produce vaccine, tool has the following advantages: due to the increase of cell growth rate and protein expression level, can boost productivity; Because do not use expensive substratum and animal cell culture systems, and reduce production cost; In addition, because do not use blood serum medium, because this increasing the safety and stability of vaccine.Whole yeast cells especially can induce extra immunne response, and yeast cells wall also can be used as the induction that adjuvant carrys out Promote immunity response.

Beneficial effect

Effectively vaccine composition can be used as according to whole yeast cells of the present invention, the various advantages of yeast can be enjoyed, make can significantly to simplify vaccine production process and (comprise lysis, or Antigen extraction, purifying and stable etc.), this produces in the process of pig circular ring virus vaccine at use recombinant microorganism is required.

Accompanying drawing explanation

Fig. 1 illustrates according to the recombinant vectors comprising the expression ORF2 of ADH1 promotor or GAL10 promotor of the present invention.

Fig. 2 illustrates the recombinant vectors of the fusion rotein of expressing Ty1 and ORF2.

Fig. 3 illustrates the result that Western blot (Westernblot) is analyzed, and carries out this western blot analysis to check the expression of ORF2 expression vector in yeast host Y2805 in Fig. 1 and Fig. 2.

Fig. 4 illustrates by the structural domain (CBD) of cellulose-binding being merged the expression vector obtained to above-mentioned carrier.

Fig. 5 illustrates the carrier by expressing Fig. 4, then carries out the result that western blot analysis obtains.

Fig. 6 illustrates by expressing the carrier of Fig. 1 and Fig. 2 and the ORF2 without His label, then the result that part that ultracentrifugation obtains carries out electrophoresis and western blot analysis is carried out, and the result of electron microscope observation, wherein carry out electron microscope observation and whether form virus-like particle (VLP) with inspection.

Fig. 7 illustrates by merging by Saccharomyces Cerevisiae in S accharomycescerevisiaeMF α and ORF2 or without the ORF2 of NLS the carrier obtained.

Fig. 8 shows the carrier by expressing Fig. 7, then carries out the result that western blot analysis obtains.

Fig. 9 illustrates the result of western blot analysis, carries out this western blot analysis to analyze the difference of expression level between various host cell of ORF2.

Figure 10 illustrate by use Y2805, BY4741, shortage gal80 Y2805 and lack the BY4741 of gal80 as host cell, and GAL10 promotor and ADH1 promotor express ORF2, then carry out the result that western blot analysis obtains.

Figure 11 illustrates the result of the substratum grown cell shown in use table 5.

Figure 12 illustrates the T/C value (test group mean value/control group mean value) that the test vaccine shown in use table 6 obtains.

Figure 13 is the graphic representation that the result that the test vaccine shown in use table 8 obtains is shown.

Figure 14 is the histogram that the result using the test vaccine shown in Figure 10 to obtain is shown.

Embodiment

Below, in further detail the present invention is described with reference to non-limiting example.That these embodiments only for illustrative object, and are not intended to limit the scope of the invention with those skilled in the art know that.

embodiment 1: prepare disease by expressing PCV2ORF2 in Saccharomyces Cerevisiae in S accharomycescerevisiae bacterial strain poison sample particle

1-1: build the carrier being used for expressing PCV2ORF2 in Saccharomyces Cerevisiae in S accharomycescerevisiae bacterial strain, and to the analysis expressed

By Bioneer synthesis for the optimized ORF2 sequence (SEQIDNO:1) of yeast codons, to carry out effective expression in yeast Saccharomycescerevisiae bacterial strain.By the gene clone of synthesis in Yeast expression carrier YEG α-HIR525.Use two kinds of promotors (GAL10 and ADH1), and by histidine-tagged for the 6-C-terminal that adds to determine the expression of ORF2.For GAL10 promotor, be structured in the primer (table 1) that 5 ' and 3 ' has EcoRI and SalI restriction enzyme recognition site in stub area, and this primer be inserted in the expression vector YEG α-HIR525 using the digestion of identical restriction enzyme.ADH1 promotor, through SmaI and EcoRI digestion, to remove GAL10 promotor, and is inserted into this position by the carrier obtained.The primer used is shown in table 1 below.Use lithium/acetate method, the recombinant expression vector obtained (Fig. 1) is transformed in Saccharomyces Cerevisiae in S accharomycescerevisiaeY2805, and yeast cell is cultivated on UD (6.7g/L yeast nitrogen, 0.77g/L lack uridylic fill-in and 20g/L glucose) flat board.Then, by PCR, transformant is screened.

table 1

Not only when for confirming the His label of expressing, and when the subunit viral vaccine of recombinating, it is known that the formation of virus-like particle increases antigenicity.When yeast, develop the technology utilizing Ty1 (long ago just known retrotransposition sub-element) in Saccharomyces Cerevisiae in S accharomycescerevisiae bacterial strain.The feature of Ty1 is, it heredity, on structure and function with retroviral nucleosides/nuclear phase seemingly.Within 1987, report the application of Ty1 in vaccine development.In addition, the known 3 ' area merges when Vaccine candidate gene and Ty1 gene and when expressing, the formation with virus-like particle is not interrupted, and virus-like particle can be formed and not need to exist the advantage of other factors in cell.Therefore, expect that Ty1 can by the formation of ORF2 helper virus sample particle.Under such expection, merge and express Ty1 (SEQIDNO:3 and SEQIDNO:4) and ORF2.Herein, merge and carry out after remove nuclear localization signal (NLS) sequence from ORF2.Increased from Y2805 with without each Ty1 of the ORF2 of NLS by PCR, so latter two PCR fragment is coupled together by over-lap PCR.In table 2 below the primer used in PCR is shown in, and be inserted in EcoRI and the SalI site of YEGa-HIR525.

table 2

In addition, use the method for lithium/acetate to be transformed in Saccharomyces Cerevisiae in S accharomycescerevisiaeY2805 by recombinant expression vector (Fig. 2), yeast cell is cultivated on UD (6.7g/L yeast nitrogen, 0.77g/L lack uridylic fill-in and 20g/L glucose) flat board.Then, by PCR, transformant is screened.

In order to check ORF2 whether effectively to express in Y2805, carry out western blot analysis after culturing.Intestinal bacteria (E.coli) to be seeded in LB substratum (1% peptone, 0.5% yeast extract and 1% sodium-chlor) and to cultivate 16 hours at 37 DEG C., single yeast colony is seeded in the UD liquid nutrient medium of 2ml meanwhile, and 30 DEG C, 180rpm carries out preculture, then mainly cultivate.For main cultivation, by YP (1%Glu and 1%Gal1) substratum (2% peptone, 1% yeast extract, 1% glucose and the 1% semi-lactosi) decile of 25ml to 250ml baffling bottle, then by pre-incubated inoculation in the medium to OD ₆₀₀reach 0.1, afterwards bacterial strain wave and culture 48 hours under 30 DEG C and 180rpm.Culture is 13, centrifugal 3 minutes of 000rpm is to remove supernatant liquor, and in culture, add 50mMTris-HCl damping fluid (pH7.0) with dissolved cell with the amount of correspond to culture 1/4, add the pearl (425 ~ 600 μm) of same volume afterwards wherein, within 5 minutes, carry out lysing cell by vortex.After lysis, centrifugal 3 minutes of 13,000rpm to remove uncracked cell and cell debris, SDS-PAGE loading dye is added in supernatant liquor, then 100 DEG C of heating 5 minutes.The material obtained places for some time on ice, is then loaded on 4 ~ 20% gradient gels, under 80V, carries out electrophoresis.Use Trans-Blot ^tMturbo (Bio-Rad) is by protein delivery on nitrocellulose filter, and this film is used in TBS-T damping fluid, and (5% skimming milk in (20mMTris-HClpH8.0,137mMNaCl, 0.1%Tween20) closes 1 hour.Film and primary antibody hatch about 1 ~ 2 hour, and use TBS-T wash buffer three times, each 5 ~ 10 minutes, then film and secondary antibody hatched 1 hour.Add the BCIP/NBT purple liquid substrate (Sigma) being conjugated to the AP (alkaline phosphatase) of secondary antibody, and hatch about 5 ~ 10 minutes with film.As a result, observe antibody binding domain and be shown as purple band.Because His label is expressed for confirming, the rabbit polyclonal IgG (SantaCruzBiotechnology) of anti-His label is as primary antibody, and the IgG alkaline phosphatase (Sigma) of goat antirabbit is as secondary antibody.In addition, the guinea pig serum through PCV2 immunity is used as primary antibody to check whether ORF2 detected.In this case, the IgG-AP (SantaCruzBiotechnology) of the anti-cavy of goat is as secondary antibody.The result of western blot analysis is shown in Figure 3.In coli strain, ORF2 weak expression when using GAL10 promotor is shown, but does not express when using ADH1 promotor.But, in Y2805, can be observed ORF2 and all express in both cases, and the expression level of ORF2 when GAL10 promotor is higher than the expression level when ADH1 promotor.In addition, illustrate when ORF2 is expressed as the fusion rotein with Ty1, express significantly a large amount of albumen.

embodiment 1-2: the amalgamation and expression of cellulose binding domain and ORF2 and analysis

When cellulose binding domain (CBD) is expressed as the fusion rotein from different albumen, it is known for can forming insoluble particle in cell.Therefore, under the expection with the function similar with virus-like particle, attempted the amalgamation and expression of CBD and ORF2.Below in two kinds of situations, attempt the amalgamation and expression with CBD: wherein α-amylase signal sequence is used for the situation of cell exocrine (SS-CBD); With, wherein do not use the situation of α-amylase signal sequence.At this, ORF2 gene is not containing nuclear localization signal (NLS).Each in amplification CBD and ORF2, then by over-lap PCR ligation amplification product, and is inserted in EcoRI and the SalI site of YEGa-HIR525.The primer used is shown in table 3 below, and is transformed in Y2805 by the plasmid (Fig. 4) built according to the method described above.

table 3

The plasmid comprising GAL10 promotor is seeded in YP (1%Glu and 1%Gal) substratum, and the plasmid comprising ADH1 promotor is seeded in YP (2%Glu) substratum, then cultivate 48 hours at 30 DEG C.When comprising the Y2805 of signal sequence, analyze cell-free extract and supernatant liquor by western blot analysis.The antibody used is the antibody of anti-His label.When there is signal sequence, can to see in cell and expression is not all observed in extracellular, this hint gene is only at cell inner expression (Fig. 5).When there is not signal sequence, observe the expression of GAL10 promotor and the expression of ADH1 promotor simultaneously, but illustrate that GAL10 promotor is more suitable for expressing in yeast.

embodiment 1-3: the ORF2 observed by expressing in Saccharomyces Cerevisiae in S accharomycescerevisiae bacterial strain forms disease poison sample particle

In the ORF2 expression cassette prepared in every way, select the yeast strain of three strains shown with efficient expression ORF2 based on Western blot.In addition, only express the pGAL10-ORF2 plasmid without His label of ORF2 according to the method preparation of embodiment 1-1, thus prepared four kinds of plasmids below: pGAL10-ORF2, pGAL10-ORF2-His, pGAL10-Ty1-ORF2 (-NLS) and pGAL-CBD-ORF2 (-NLS)-His.The Y2805 with each plasmid is cultivated 48 hours with the condition of 30 DEG C and 180rpm in YPDG.Each culture all carrys out collecting cell in centrifugal 10 minutes at 13,000rpm.In cell, add TEN damping fluid (10mMTris-HClpH7.4,2mMEDTA, 140mMNaCl) and pearl, then vortex about 10 minutes is with lysing cell.Within centrifugal 20 minutes at 13,000 and 4 DEG C, to collect supernatant liquor, the supernatant liquor then collected carries out ultracentrifugation to cell pyrolysis liquid.By ultracentrifugation, obtain the sucrose solution of in TEN damping fluid 15%, 25%, 35%, 45% and 60%, and be sequentially poured in centrifuge tube to form saccharose gradient.The cell-free extract prepared as mentioned above is added to the top of centrifuge tube and at 36,000rpm centrifugal 4 hours.Gradient is divided into the decile of 1.5-ml from the bottom of pipe, then carries out western blot analysis.Pass through Western blot, collect the component that ORF2 band is only shown, use ultra-filtration centrifuge tube (Amiconultracentrifugalfilter, Millipore, 10K film) from this component, remove sucrose, concentrate this component afterwards and use electron microscope to observe.When only expressing ORF2, observe the virus-like particle (Fig. 6) defining and there is about 25nm size.But, when there is His label, not forming virus-like particle, showing the formation of the His label meeting viral interference sample particle be combined with C-end.In addition, the formation of expection helper virus sample granuloplastic Ty1 viral interference sample particle is slightly shown.When ORF2 is expressed as the fusion rotein with CBD, there is His label although observe, define virus-like particle well.

the secreting, expressing of embodiment 2:ORF2 and analysis

The advantage of recombinant subunit vaccine is, if its extracellular secreting, expressing if possible, then eliminates the demand to lysis and easy purifying.Generally, except several other advantage (such as security), different from intestinal bacteria, yeast is suitable for carrying out secreting, expressing because defining its protein excretion mechanism.Therefore, the secreting, expressing carrying out ORF2 in yeast is attempted.

When expression in escherichia coli full-length gene, its expression level is very low.But report: be not that required this is true based on the 47-aminoacid sequence being positioned at aminoterminal NLS region for the formation of conformational epitope, the clone expressed without NLS increases its expression level.For the secretion of ORF2, use Saccharomyces Cerevisiae in S accharomycescerevisia ethe signal sequence of mating factor α (matingfactoralpha, MF α).Construct the carrier of expressing the full length sequence of ORF2 and the sequence without NLS of ORF2.The primer used is shown in table 4 below.Use and carry out optimized composition sequence as template, by pcr amplification gene for yeast codons.Use XbaI and SalI digest amplification product, and be inserted in XbaI and the SalI site of YEG α-HIR525.The schematic diagram of the carrier built is shown in Figure 7.Use lithium/acetate method, by each vector to Saccharomyces Cerevisiae in S accharomycescerevisia ein Y2805.Yeast cell is cultivated on UD (6.7g/L yeast nitrogen, 0.77g/L lack uridylic fill-in and 20g/L glucose), then by PCR, transformant is screened.

table 4

Cell is carried out preculture in UD liquid nutrient medium, then cultivates 48 hours in YP (1%Glu and 1%Gal) substratum and use anti-His antibody to carry out western blot analysis.When there is MF α secretion signal, no matter whether there is NLS (Fig. 8), ORF2 is neither secreted in substratum also not at cells.In addition, when there is not NLS, the expression level of ORF2 is significantly increased.

embodiment 3: select the bacterial strain being used for expressing ORF2 in Saccharomyces Cerevisiae in S accharomycescerevisiae bacterial strain

Being used in embodiment 1-1 the GAL10 promotor observed and high expression level is shown, ORF2 being incorporated into through being usually used in recombinant expressed multiple Saccharomyces Cerevisiae in S accharomycescerevisia ein bacterial strain, and contrast the expression level of the ORF2 between bacterial strain.As expression vector, use the pGAL10-ORF2 built in embodiment 1-3.For with Saccharomyces Cerevisiae in S accharomycescerevisia ethe bacterial strain that Y2805 carries out contrasting is INVSc1, ATCC200589, BY4741, L3262, ATCC201228 and ATCC201741.To carry out transforming and culturing process with described identical mode above, and by western blot analysis, the sample with amount is analyzed.Analytical results is shown in Figure 9.Make the expression level of optical density 1D program (UVIBandMax) comparative analysis ORF2.ORF2 does not express in ATCC200589 bacterial strain, and expression in ATCC201228 is not remarkable yet.In addition, when the expression level of ORF2 in Y2805 bacterial strain is counted as 100, the expression level of ORF2 in residue bacterial strain is 19.7% in INVSc1, and be 15.0% in BY4741, be 40.0% in L3262, and be 20.4% in ATCC201741.As mentioned above, can see that the expression level of ORF2 is significantly different between bacterial strain, and the highest in Y2805 bacterial strain.

Observe in the situation of GAL10 promotor of high expression level in the above-described embodiments, in order to the optimization expression of the promotor of semi-lactosi induction, should meet in substratum and there is not glucose but there is semi-lactosi.But compared with glucose, semi-lactosi is very expensive carbon source.When developing the semi-lactosi even not adding costliness when only using glucose before the present inventor, also can use the Saccharomyces Cerevisiae in S accharomycescerevisia lacking gal80 ebacterial strain economically Restruction albumen method (Korean patent No. 10-0947376 (on March 5th, 2010), exercise question be " a kind of use lack GAL80 yeast strain Restruction albumen method ( gAL80 ) ").

In order to check the expression pattern of ORF2 in the bacterial strain lacking gal80, lithium/acetate method is used to be transformed in following bacterial strain by the pGAL10-ORF2 built in embodiment 1-3: the Saccharomyces Cerevisiae in S accharomycescerevisia that most high expression level is shown as mentioned above ey2805 Δ gal80, illustrates the BY4741 of low expression level, and BY4741 Δ gal80.By strain culturing on UD (6.7g/L yeast nitrogen, 0.77g/L lack uridylic fill-in and 20g/L glucose) flat board, then by PCR, transformant is screened.When yeast, single yeast colony is seeded in the UD liquid nutrient medium of 2ml, under 30 DEG C and 180rpm, carries out preculture, then carry out main cultivation.For main cultivation, when Y2805 and BY4741,25mlYP (1%Glu1 and 1%Gal) substratum (2% peptone, 1% yeast extract, 1% glucose and 1% semi-lactosi) is added in 250ml baffling bottle, when Y2805 Δ gal80 and BY4741 Δ gal80,25mlYP (2%Glu) is added in 250ml baffling bottle, then by the inoculation of each initial incubation to substratum until OD ₆₀₀reach 0.1.The bacterial strain of inoculation shakes cultivation 48 hours under 30 DEG C and 180rpm.The bacterial strain cultivated carries out western blot analysis in the same manner as described above, and analytical results is shown in Figure 10.Can observe, ORF2 expresses in four kinds of bacterial strains, but the expression level of ORF2 in Y2805 bacterial strain is significantly higher than the expression level in BY4741 bacterial strain, and the expression level in GAL10 promotor is higher than the expression level (Figure 10) in ADH1 promotor.

embodiment 4: the fermentor cultivation expressing the recombinant Saccharomyces cerevisiae Saccharomycescerevisiae bacterial strain of ORF2

For the animal testing using circovirus virus vaccine to carry out in pig, the restructuring yeast strains of expressing ORF2 is cultivated in fermentor tank.Batch feed is cultivated in 5L fermentor tank to observe the pGAL10-ORF2/Y2805 Δ gal80 bacterial strain (that is, the combination of gene expression plasmid and yeast host cell) that most high expression level is shown in the above-described embodiments.Table 5 below shows the substratum composition used in batch feed is cultivated.

table 5

Use the ammoniacal liquor of 24% that the culture of batch feed is adjusted to pH6.0, culture temperature is maintained 30 DEG C.Cell grows gradually, until OD ₆₀₀reach 71 (Figure 11).After culturing, harvested cell and be used in 1, the homogenizer under 000 bar carries out four cracking, prepares cell pyrolysis liquid according to the method described in embodiment 1.

embodiment 5: immunogenic research

embodiment 5-1: the test vaccine of preparation various ways (is included in Saccharomyces Cerevisiae in S accharomycescerevisiae bacterial strain the ORF2 of middle expression) and use cavy to carry out immunogenic research

In order to evaluate at yeast saccharomyces cerevisiae saccharomycescerevisiaewhether the ORF2 expressed in bacterial strain can be used as antigen, ORF2 is expelled in cavy body whether produce antibody with inspection.At this, according to presence or absence cell pyrolysis liquid, presence or absence is used for the His label of purifying, or presence or absence NLS, carries out comparative evaluation.Particularly, in order to overcome the deficiency that yeast cells wall is difficult to destroy, and whether can use immediately when not carrying out lysis to check the full cell of the yeast strain of expressing recombinant protein, use the full cell observing the yeast strain of expressing ORF2 to prepare test vaccine, and the vaccine of preparation is divided into hot deactivation group and Formalin inactivation group.

As described in example 1 above culture expression ORF2 Y2805 Δ gal80 bacterial strain and carry out centrifugal, and based on OD ₆₀₀value (1OD=2 × 10 ⁷/ ml) estimate the quantity of full yeast cell that obtains.Use full yeast cell, the concentration according to cell prepares test vaccine, with 2 × 10 ⁹the concentration of/ml is inoculated.Two kinds of methods are used for carrying out deactivation to full yeast cell.For hot deactivation, cell processes 2 hours at 60 DEG C, then stores until use at-20 DEG C.For Formalin inactivation, cell uses the formalin of 0.2% room temperature treatment 72 hours, is then coated on substratum to confirm that yeast cell does not grow.

In addition, in order to obtain the ORF2 of cell inner expression from above-mentioned cell, with described identical mode lysing cell in embodiment 1, thus prepare cell pyrolysis liquid.In addition, following four kinds of test vaccine have also been prepared: ORF2 cell pyrolysis liquid; By the vaccine (ORF2 (MF)) that the membrane filter cell pyrolysis liquid of 0.2 micron obtains to remove cell debris; The vaccine (ORF2 (-NLS)) obtained by removing 47-amino acid N LS sequence from the N-terminal of ORF2; With, the vaccine (ORF2 (+His)) obtained by adding His label at the C-terminal of ORF2.About the information of test vaccine shown in table 6, what comprise preparation comprises the test vaccine of adjuvant and the type of vaccine composition and injection volume.

table 6

Use the PCV2 vaccine CircoFLEX (BoehringerIngelheim) that market is sold as positive control, the immunogenicity of inspection antigen candidate thing.Preparation has the test vaccine of the composition identical with positive control.Test vaccine is inoculated in cavy body, then carries out blood collecting and be separated with serum.The serum obtained carries out ELISA, to contrast immunogenicity.

Use cavy as test animal, the one in every cavy injection 1ml six kinds of test vaccine.Use CircoFLEX (BoehringerIngelheim) as positive control, use PBS as negative control.For injection, each vaccine is subcutaneously injected into and is made up of in the animal body of a group 8 animals.After three weeks, collect the blood from these animals, and from blood, isolate serum and 56 DEG C of deactivations 30 minutes, then carry out ELISA.For ELISA, use 1 μ g/ml monoclonal antibody (purchased from JBT) to carry out Ab and detect sandwich indirect ELISA, and as PCV2ORF2 antigen, the undiluted antigen purchased from BoehringerIngelheim uses after carrying out 20 times of dilutions.Serum sample uses after carrying out 50 times of dilutions.Use being coated on 37 DEG C and carrying out 2 hours of PCV2 specific antibody, be enclosed in 37 DEG C and carry out 2 hours.In addition, use being coated on 37 DEG C and carrying out 1 hour of PCV2 antigen, detection reaction carries out 1 hour at 37 DEG C.Conjugation reaction carries out 1 hour at 37 DEG C, substrate under dark condition room temperature reaction 10 minutes.The value (table 7) of O.D450 is measured after adding stop bath.

table 7

T/C value (mean value of the mean value/control group of test group) in table 7 is shown in Figure 12.The T/C value of 2.0 or higher is judged as the positive.When contrasting mean value, test vaccine 1,2 and 3 illustrates the result substantially similar with positive control, and this implies that they are positive as recombiant vaccine antigen; Wherein test vaccine 1 and 2 comprises whole yeast cells, the cell pyrolysis liquid of what test vaccine 3 obtained after being lysis comprise cell walls.Such result is consistent with result of study before, shows that whole yeast cells component plays the function of adjuvant.When using full cell, the pretreatment process comprising hot deactivation and Formalin inactivation does not have difference in immunogenicity.Removed by membrane filter wherein in the situation of MF of cell debris, lower slightly immunogenicity is seemingly because injection volume is lower slightly, and when comprising the test vaccine being with the fusion rotein of His label as antigen, show the combination that the steric hindrance brought by His label can affect antibody.Meanwhile, the ORF2 (-NLS) lacking N-terminal NLS illustrates negative value, and this shows that it is not suitable as vaccine candidate object, and these are different from the report that NLS does not have remarkably influenced to immunogenicity.

embodiment 5-2: be used in the ORF2 target of expressing in Saccharomyces Cerevisiae in S accharomycescerevisiae bacterial strain and determine piglet pair immunogenic research

Prepare the test vaccine that three kinds comprise PCV2ORF2, and use the fixed pig of circovirus virus vaccine target to carry out the immunogenicity of verification test vaccine as test animal.As shown in table 8, prepare the full cell of the recombination yeast prepared according to the method for embodiment 5-1, cell pyrolysis liquid and through (MF) of membrane filtration as test vaccine, and to inoculate.

table 8

In animal testing, by each vaccine intramuscularly 2ml to the pig body often organized once, often organizes and be all made up of the pig in three 10 ~ 12 week ages, and animal testing carries out 6 weeks.As positive control, intramuscularly 1mlCircoFLEX.Measured the change of the immunogenic S/P value of instruction by single ELISA (symphysis unit (Synbiotics)) of closing, measuring result is shown in table 9 below.The mean value of S/P value is shown in Figure 13.The PCVORF2 expressed in yeast is considered to the effect playing vaccine, because it illustrates the change similar with CircoFLEX.Particularly, illustrate that antibody titers is more and more higher with the order of carrying out the test vaccine of MF process, cell pyrolysis liquid and full cell, and the highest in whole-cell vaccines.

table 9

embodiment 6: the inspection of the dosetest of whole yeast cells and the possibility without Adjuvanted vaccines

When yeast, beta-glucan (cell-wall component) can be used as adjuvant come activate immunity response be known.Therefore, in the present invention, examine whole yeast cells whether to can be used as without Adjuvanted vaccines.In addition, whether the ORF2 (ORF2-Ty1) examined containing Ty1 also can be used as without Adjuvanted vaccines, and wherein known Ty1 contributes to the formation of virus-like particle.

Particularly, the whole yeast cells of expressing ORF2 is divided into the group containing adjuvant and the group without adjuvant, and the whole yeast cells of expressing ORF2-Ty1 fusion rotein is used as without Adjuvanted vaccines, is prepared as follows the test vaccine shown in table 10 in face thus.Each test vaccine be all expelled in cavy body, the serum of cavy carries out ELISA to evaluate the immunogenicity of each vaccine.

table 10

When without Adjuvanted vaccines, examining compared with traditional dose, whether can maintain the immunogenicity of vaccine when reducing dosage.The amount of the whole yeast cells measured in above-mentioned animal testing is 2 × 10 ⁹/ ml, whole yeast cells is diluted 10 times and 100 times respectively to 2 × 10 ⁸/ ml and 2 × 10 ⁷/ ml, then tests.When the group without adjuvant, whole yeast cells is with 2 × 10 ⁹the concentration of/ml is tested.Whole yeast cells carries out deactivation in 1 week by using 0.2% formalin process.As adjuvant, use the aluminum hydroxide gel of 20% concentration.

In animal testing, the one in every cavy injection 1ml five kinds of test vaccine.Use CircoFLEX (BoehringerIngelheim) as positive control, and use the group of not injecting as negative control.Altogether test 7 groups, each group is all made up of 10 animals.By intramuscularly, by each vaccination in the body of each treated animal, and collect the blood from 5 animals at the 3rd week, collect the blood from residue 5 animals at the 5th week.From the blood collected, isolate serum, 56 DEG C of deactivations 30 minutes, then carry out ELISA.ELISA detects sandwich indirect ELISA by Ab to carry out, and test result is shown in table 11 below, table 12 and Figure 14.

table 11

	Serum 1	Serum 2	Serum 3	Serum 4	Serum 5	Average OD	T/C value	Judge
									Vac 1	3.437	3.354	3.250	3.173	3.437	3.330	7.00	Positive
Vac 2	3.241	3.212	2.924	3.172	3.206	3.151	6.63	Positive
									Vac 3	2.964	3.183	3.041	3.260	3.255	3.141	6.60	Positive
Vac 4	3.310	3.194	3.215	3.222	3.283	3.245	6.82	Positive
									Vac 5	3.390	3.468	3.407	2.907	3.440	3.322	6.99	Positive
Positive control	2.886	3.305	3.001	2.846	2.976	3.003	6.32	Positive
									Negative control	0.449	0.502				0.476

*, when T/C value is 2.0 or higher, the positive is judged as.

table 12

	Serum 1	Serum 2	Serum 3	Serum 4	Serum 5	Average OD	T/C value	Judge
									Vac 1	3.444	3.445	3.394	3.346	3.374	3.401	8.49	Positive
Vac 2	3.416	3.220	3.430	3.421	3.078	3.313	8.27	Positive
									Vac 3	3.331	3.202	3.410	3.333	3.318	3.319	8.29	Positive
Vac 4	3.419	3.501	3.462	3.469	3.212	3.413	8.52	Positive
									Vac 5	2.763	2.462	2.760	2.413		2.600	6.49	Positive
Positive control	3.259	3.131	3.027	3.206	2.976	3.118	7.79	Positive
									Negative control	0.354	0.447				0.401

The result being detected sandwich indirect ELISA serum analysis by Ab is shown, express PCVORF2 whole yeast cells containing in adjuvant group (vac1 ~ vac3), at 2x10 ⁷individual cell ~ 2x10 ⁹the dosage range endoantigen amount of individual cell (cfu) does not have difference, and this positive T/C value showing that these are organized is equal to or higher than the T/C value of positive control.In addition, the immunogenicity without adjuvant group (vac4) of whole yeast cells expressing PCVORF2 equals the immunogenicity of positive control substantially, and whether this need to check can even 2 × 10 if showing ⁹immunogenicity is maintained under individual cell or lower dosage.

Therefore, can find out, when the whole yeast cells of expressing PCVORF2 uses together with adjuvant, give at least 2 × 10 ⁷the above-mentioned cell of the amount of individual cell is just shown with vaccine effect.It can also be seen that, even if when not adding independent adjuvant (without adjuvant), cell also illustrates vaccine effect.

In addition, in the level (2 × 10 similar with other test group ⁹individual cell) under, the whole yeast cells (vac5) of expressing ORF2-Ty1 fusion rotein is also positive.

Sequence list text none

SEQIDNO.1: the nucleotide sequence carrying out optimized ORF2 for yeast codons synthesized by Bioneer, with at yeast saccharomyces cerevisiae saccharomycescerevisiaemiddle effective expression ORF2;

SEQIDNO.2: the aminoacid sequence carrying out optimized ORF2 for yeast codons, with at yeast saccharomyces cerevisiae saccharomycescerevisiaemiddle effective expression ORF2;

SEQIDNO.3: yeast saccharomyces cerevisiae saccharomycescerevisiaethe nucleotide sequence of retrotransposon elements T y1;

SEQIDNO.4: yeast saccharomyces cerevisiae saccharomycescerevisiaethe aminoacid sequence of retrotransposon elements T y1.

The Y2805 bacterial strain (Y2805/pGAL10-Ty1-ORF2) of expressing the Y2805gal80 deletion mycopremna (Y2805 △ gal80/pGAL10-ORF2) of ORF2 and the fusion rotein of expression Ty1 and ORF2 is deposited in Korea S's Culture Collection (KCTC) on February 20th, 2013, and registration number is respectively KCTC12372BP and KCTC12373BP.

The title of depositary institution: Korea Institute of Bioengineering

Registration number: KCTC12372BP

Preservation date: on February 20th, 2013

The title of depositary institution: Korea Institute of Bioengineering

Registration number: KCTC12373BP

Preservation date: on February 20th, 2013

PCT/RO/134 shows

Claims

1., for a whole yeast cells for pig circular ring virus vaccine, described whole yeast cells comprises the II type capsid Orf2 gene of pig circular ring virus.

2. whole yeast cells according to claim 1, wherein, described Orf2 gene is represented by SEQIDNO:1.

3. whole yeast cells according to claim 1, wherein, the albumen that described Orf2 genes encoding is represented by SEQIDNO:2.

4. whole yeast cells according to claim 1, wherein, yeast is used as host cell and is the bacterial strain of Saccharomyces Cerevisiae in S accharomycescerevisiae.

5. whole yeast cells according to claim 4, wherein, described bacterial strain is Y2805 bacterial strain.

6. whole yeast cells according to claim 1, wherein, described whole yeast cells is through heat-inactivated cell or the cell through Formalin inactivation.

7., for a lysate for the whole yeast cells of pig circular ring virus vaccine, described lysate comprises the II type capsid Orf2 gene of pig circular ring virus.

8. the lysate of whole yeast cells according to claim 7, wherein, described Orf2 gene is represented by SEQIDNO:1.

9. the lysate of whole yeast cells according to claim 7, wherein, the albumen that described Orf2 genes encoding is represented by SEQIDNO:2.

10. the lysate of whole yeast cells according to claim 7, wherein, yeast is used as host cell and is the bacterial strain of Saccharomyces Cerevisiae in S accharomycescerevisiae.

The lysate of 11. whole yeast cells according to claim 10, wherein, described bacterial strain is Y2805 bacterial strain.

12. 1 kinds of pig circular ring virus vaccine compositions, comprise the lysate of whole yeast cells or this cell, and this whole yeast cells comprises the II type capsid protein ORF2 of pig circular ring virus.

13. pig circular ring virus vaccine compositions according to claim 12, wherein, the genes encoding that described albumen ORF2 is represented by SEQIDNO:1.

14. pig circular ring virus vaccine compositions according to claim 12, wherein, described albumen ORF2 is the polypeptide represented by SEQIDNO:2.

15. pig circular ring virus vaccine compositions according to claim 12, wherein, yeast is used as host cell and is the bacterial strain of Saccharomyces Cerevisiae in S accharomycescerevisiae.

16. pig circular ring virus vaccine compositions according to claim 15, wherein, described bacterial strain is Y2805 bacterial strain.

17. pig circular ring virus vaccine compositions according to claim 16, wherein, described Y2805 bacterial strain lacks gal80 gene.

18. pig circular ring virus vaccine compositions according to claim 12, wherein, described whole yeast cells is through heat-inactivated cell or the cell through Formalin inactivation.

19. pig circular ring virus vaccine compositions according to claim 12, described pig circular ring virus vaccine composition is without adjuvant.

20. according to claim 12 to the pig circular ring virus vaccine composition according to any one of 19, and wherein, described whole yeast cells is with 4 × 10 ⁹individual cell or lower concentration use.

21. 1 kinds of productions are used for the method for the whole yeast cells through transforming of pig circular ring virus vaccine, said method comprising the steps of:

Preparation comprises the recombinant expression vector of Orf2 gene, the II type capsid protein of this Orf2 genes encoding pig circular ring virus; With

Use recombinant vectors transformed yeast.

22. methods according to claim 21, wherein, described Orf2 gene is the gene represented by SEQIDNO:1.

23. methods according to claim 21, wherein, the polypeptide that described Orf2 genes encoding is represented by SEQIDNO:2.

24. methods according to claim 21, wherein, described yeast is used as host cell and is the bacterial strain of Saccharomyces Cerevisiae in S accharomycescerevisiae.

25. methods according to claim 24, wherein, described bacterial strain is Y2805 bacterial strain.

26. methods according to claim 25, wherein, described Y2805 bacterial strain lacks gal80 gene.

27. methods according to claim 21, wherein, described recombinant expression vector comprises GAL10 promotor.

28. methods according to claim 21, wherein, described Orf2 gene and Ty1 merge, to increase the expression of described Orf2 gene.

29. methods according to claim 21, comprise further and carry out heat-inactivated step to described whole yeast cells.

30. methods according to claim 21, comprise the step of described whole yeast cells being carried out to Formalin inactivation further.

31. 1 kinds of productions are used for the method for the whole yeast cells lysate through transforming of pig circular ring virus vaccine, said method comprising the steps of:

Preparation comprises the recombinant expression vector of Orf2 gene, the II type capsid protein of this Orf2 genes encoding pig circular ring virus;

Use recombinant vectors transformed yeast to prepare transformant; With

Transformant described in cracking.

32. 1 kinds is the vaccinated method of pig, comprises the lysate giving described pig whole yeast cells or this cell, and this whole yeast cells comprises the II type capsid protein ORF2 of pig circular ring virus.

33. methods according to claim 32, wherein, the genes encoding that described ORF2 is represented by SEQIDNO:1.

34. methods according to claim 32, wherein, described ORF2 is the polypeptide represented by SEQIDNO:2.

35. methods according to claim 32, wherein, described yeast is used as host cell and is the bacterial strain of Saccharomyces Cerevisiae in S accharomycescerevisiae.

36. methods according to claim 35, wherein, described bacterial strain is Y2805 bacterial strain.

37. methods according to claim 36, wherein, described Y2805 bacterial strain lacks gal80 gene.

38. methods according to claim 32, wherein, described recombinant expression vector comprises GAL10 promotor.

39. methods according to claim 32, wherein, described ORF2 and Ty1 merges, to increase the expression of described ORF2.