CN102363751A - Dengue virus (DENV)-like particle as well as preparation method and application thereof - Google Patents
Dengue virus (DENV)-like particle as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a dengue virus (DENV)-like particle as well as a preparation method and the application thereof. P. pastoris yeast strains transformed by recombinant carriers of virus-like particles (VLPs) of DENV1-4, which are respectively pGAPZ-Alpha-PrME-D1-X33, pGAPZ-sPrME472-D2-X33, pGAPZ-sPrME-D3-X33 and pGAPZ-sPrME-D4-X33, are respectively preserved at China center for type culture collection (CCTCC) at Wuhan University on March 14th, 2011 and have the serial numbers of CCTCCM2011063, CCTCCM2011064, CCTCCM2011065 and CCTCCM2011066. The VLPs of the DENV1-4 are obtained by transfecting the recombinant carriers stated in claim 1 with P. pastoris yeast cells X33 and enabling the recombinant carriers to secrete the VLPs. According to the preparation method, a P. pastoris yeast system not relied on carbinol is adopted to prepare the DENV-like particle, which is not reported at home and abroad yet, so the innovativeness is achieved. The DENV-like particle prepared from the system can be used for preparing vaccines, and various defects in the research of dengue vaccines such as attenuated live vaccines, subunit vaccines, carrier vaccines and the like are overcome; and the DENV-like particle has the advantages of safety and high efficiency, and is suitable for large scale production.
Description
Technical field
The present invention relates to the genetically engineered field, particular content prepares dengue virus appearance particulate method and application for using pichia spp.
Background technology
Dengue virus (dengue virus; DENV) with the yellow-fever mosquito be communication media, infected person can cause singapore hemorrhagic fever (dengue fever, DF), dengue hemorrhagic fever (dengue hemorrhagic fever; DHF) and step on leather shock disease (dengue shock syndrome, DSS).DHF/DSS is in a bad way, and case fatality rate is high, and its pathogenesis is not illustrated so far as yet.Owing to lack effective dengue virus vaccine, add control mosquito matchmaker's practical difficulty, dengue virus infection popular region and epidemic strength are constantly being enlarged.Singapore hemorrhagic fever stages a comeback in many places in the whole world at present, has become the widest, that morbidity is maximum, harm the is bigger a kind of arthropod borne viral disease that distributes in the world.Therefore, strengthen the research of DENV mechanism of causing a disease, vaccine and the development of quick diagnosis reagent kit and become very urgent problem.
The research of stepping on the leather vaccine has had the history of last 100 years, but also still manque at present vaccine comes out.The factor that the leather vaccine research is stepped in restriction mainly contains the following aspects: (1) lacks animal model suitable, that can simulate human infection DENV clinical symptom; (2) infection causes the concrete mechanism of DHF and DSS it be unclear that to DENV; (3) exist between the DENV different shaped infection enhancement that antibody relies on (antibody-dependent enhancement, ADE).Therefore developing the DENV vaccine need all have the isostatic provide protection to 4 types, has therefore increased the difficulty of development.
Virus-like particle (Virus-like particles, VLPs) vaccine appear as research and development new type of safe efficiently vaccine a new opportunity is provided.VLPs is meant what the one or more structural protein by virus were assembled into, does not contain viral nucleic acid, can not carry out the hollow bead of self-replicating.Because VLPs does not comprise viral DNA or RNA, therefore there are not the answer of virulence and the hidden danger of homologous recombination.Simultaneously, VLPs has kept identical with the natural viral particle or similar sterie configuration and the epitope of inducing neutralizing antibody, has very strong immunogenicity.Not only the generation humoral immunization can be induced, and CD4 can be excited
+The propagation of T cell and ctl response (cytotoxic T lymphocyte).The VLPs of most viruses realizes effectively oneself's assembling through the vivoexpression system at present, comprising HIV, and Influenza A virus, Hantaan virus, Rotavirus, viruses such as Papillomavirus.
Dengue virus is the member of flaviviridae Flavivirus, is the sub-thread positive chain RNA virus, and genome is about 11kb, contains a single opening code-reading frame.Sequence in the gene is 5'-NCR-C-PrM/M-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-NCR-3' in proper order, encode altogether 3 structural protein and 7 Nonstructural Proteins.Wherein the E albumen of E genes encoding is the virus surface envelope glycoprotein, has with cell surface receptor to combine, and the mediation film merges, and induces various biological functions such as producing neutralizing antibody, is viral most important antigen composition.The PrM/M albumen of PrM genes encoding also is considered to induce the antigenic component that produces neutralizing antibody.Nonstructural gene and non-coding region are main relevant with virus replication and protein translation.Report; The envelope E protein of flavivirus and prM albumen are in the vivoexpression system during coexpression; The polymer particle that is foldable to similar natural viral particle space structure is a virus-like particle, be also referred to as subviral particle (subviral particles, SVPs).Aspect dengue virus; Have investigator's using yeast system, CHO-K1 cell etc. to carry out the trial that VLPs expresses, but low expression level and the maintenance of protein function of DENV VLPs in mammalian cell is to hinder the bottleneck of stepping on the development of leather VLPs vaccine always.Therefore, select suitable DENV VLPs expression system, and take the reasonably tactful VLPs of raising expression output and secretion level to become development DENV VLPs vaccine urgent problem.At present, do not find to have the report of four type dengue viruss of secreting, expressing virus-like particle research in the pichia spp eukaryotic system in the correlative study as yet.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, dengue virus 1-4 is provided virus-like particle.
To achieve these goals, the present invention adopts following technical scheme:
The present invention at first makes up and is used for the primer sequence that different recombinant expressed son adopts, the dengue virus SprME gene that can increase respectively, and its length is 1983 bp, coding prM signal peptide, the E albumen of prM albumen and total length.What can increase again is the SprME472 gene, and its length is 1914 bp, coding prM signal peptide, the E albumen that prM albumen and TM2 end block.
The present invention also is provided for expressing the gene element of DENV1～4 virus-like particles.It is to adopt the RT-PCR technology to obtain the prM-E protein coding gene sequence of DENV-1 GZ01/95 strain, DENV-2 ZS01/01 strain, DENV-3 H87 strain and DENV-4 H241 strain.In order to make dengue virus VLPs can obtain to efficiently express; We transform 1～4 type dengue virus CprM-E or prME expression of gene element; Be included in the signal peptide sequence of prM gene N end design ideal; Replace the portion gene of four type dengue virus E gene anchorage zones etc., the present invention makes up and has expressed the prME472 that clips E albumen TM2 (E protein 47 2aa-496aa) end and contains the proteic prME of total length E, has finally confirmed to express the best construction strategy of VLPs.Four type dengue virus prM-E gene elements are called after: DENV1-PrME respectively, DENV2-sPrME472, DENV3-sPrME, DENV4-sPrME.
The present invention also provides can four type dengue viruss of secreting, expressing appearance particulate P.pastoris yeast strain.It is through special restriction enzyme site Bsp119I and Xba I, respectively four above-mentioned type dengue virus prM-E gene elements is cloned among the eukaryotic system expression vector pGAPZ α A.Difference called after: I type dengue virus appearance particle Pichia anomala expression engineering bacteria pGAPZ-α-PrMED1-X33, II type dengue virus appearance particle Pichia anomala expression engineering bacteria pGAPZ-sPrME472-D2-X33, III type dengue virus appearance particle Pichia anomala expression engineering bacteria pGAPZ-sPrME-D3-X33 familial combined hyperlipidemia dengue virus appearance particle Pichia anomala expression engineering bacteria pGAPZ-sPrME-D4-X33; Again recombinant vectors is transformed in the P.pastoris yeast X33; Obtain the engineering bacteria of stably express dengue virus virus-like particle; Respectively at being preserved in Wuhan University China typical culture collection center on March 14th, 2011, numbering is respectively CCTCC M 2011063, CCTCC M 2011064, CCTCC M 2011065 and CCTCC M 2011066.
The present invention also provides the dengue virus appearance particulate preparation method of said gene coding, and its step comprises:
1) adopt the RT-PCR technology from DENV-1 GZ01/95 strain, DENV-2 ZS01/01 strain, DENV-3 H87 strain and DENV-4 H241 strain, to obtain the prM-E gene element of four type dengue viruss respectively;
2) 1～4 type dengue virus CprM-E or prME expression of gene element are transformed, be included in the signal peptide sequence of prM gene N end design ideal, replace the portion gene of four type dengue virus E gene anchorage zones;
3) the prM-E genetic modification element with four type dengue viruss is cloned into respectively in the eukaryotic system expression vector;
4) with 3) in the transfection respectively of the recombinant expression vector that obtains
P.pastorisYeast X33 cell, and make its secreting, expressing dengue virus virus-like particle;
5) with SDS-PAGE, Western blot technology, testing goal genetic expression.
6), detect the morphological specificity of virus-like particle with 15-60% SDGC and electron microscopy.
The present invention further provides the dengue virus appearance particle of said gene coding to step on the application in the leather type disease in prevention.The present invention uses DENV1～4-VLPs unit price, two valency and tetravalence compatibility immunity Balb/c mouse respectively, can bring out neutralizing antibody, cellular immunization and immunological memory reaction, for the dengue virus vaccine development is laid a good foundation.
Compared with prior art, the present invention adopts following technical scheme:
The present invention adopts non-methyl alcohol to rely on
P .pastorisYeast system prepares dengue virus appearance particle, does not appear in the newspapers as yet both at home and abroad, has novelty.The dengue virus appearance particle of this systems produce can be used to prepare vaccine, has both overcome present attenuated live vaccine, subunit vaccine and carrier bacterin etc. and has stepped on the many shortcomings in the leather vaccine research, has safe and efficient, as to be suitable for large-scale production advantage.
Description of drawings
Fig. 1 is a pGAPZ α A expression vector structural representation used in the present invention, and this carrier comprises the GAP promotor of composing type, coding yeast saccharomyces cerevisiae signal peptide gene sequence and the antibiotic gene of the anti-Zeocin of coding.
Fig. 2 is a construction of recombinant vector synoptic diagram of the present invention, A: dengue virus envelope protein prM and E.The alpha-helix zone at the terminal stem of the E PROTEIN C position of H1 (400-413 aa) and H2 (430-449 aa) representative prediction.CS (413-430 aa) represents the zone of interval H1 and H2 spiral.The E albumen of TM1 (449-472 aa) and TM2 (473-495 aa) representative prediction is striden diaphragm area.The cleavage site of arrow representation signal peptase.B: the structure of different recombinant expression vectors.P
GAPRepresent the GAP promotor among the pGAPZ α A.S represents the signal peptide sequence of prM, and α represents the signal peptide sequence among the pGAPZ α A.
Fig. 3 is that the recombinant vectors yeast transformant that makes up of the present invention is with GAP universal primer PCR qualification result; Wherein swimming lane 1 is the contrast of empty carrier transformant; 2～4th, the pGAPZ-sPrME472 transformant; The 5th, yeast X33 contrast, 6～9th, pGAPZ-sPrME transformant, the 10, the 11st, pGAPaZ-sPrME transformant.
Fig. 4 be the recombinant vectors yeast transformant that makes up of the present invention with goal gene special primer PCR qualification result, wherein swimming lane 2～4th, the pGAPZ-sPrME transformant.
Fig. 5 also be the recombinant vectors yeast transformant that makes up of the present invention with goal gene special primer PCR qualification result, wherein swimming lane 2～4th, the pGAPZ-sPrME472 transformant.
Fig. 6 is the Western blot evaluation figure that VLPs of the present invention expresses in yeast cell; Wherein 1,2 is respectively the Western blot qualification result of pGAPaZ-PrME transformant express cell lysate and expression supernatant and the reaction of dengue virus specific monoclonal antibody; 3,4 is respectively the Western blot qualification result of pGAPZ-sPrME472 transformant express cell lysate and expression supernatant and the reaction of dengue virus specific monoclonal antibody; 5,6 is respectively the Western blot qualification result of pGAPZ-sPrME transformant express cell lysate and expression supernatant and the reaction of dengue virus specific monoclonal antibody, the visible E albumen and the 20 kDa prM albumen that all can detect about 50 kDa at the cell pyrolysis liquid and the fermentation supernatant of each recon.
Fig. 7 is the Western blot evaluation figure of the present invention with the VLPs of sucrose density gradient centrifugation purifying expression; A1～3rd wherein, the pGAPaZ-PrME transformant is expressed VLPs and is collected at 30%, 35%, 40% sucrose layer; B1～3rd, the pGAPZ-sPrME transformant is expressed VLPs and is collected at 30%, 35%, 40% sucrose layer, and the dengue virus VLPs that visible the present invention expresses contains E, PrM and M albumen.
Fig. 8 is the negative staining electromicroscopic photograph of the VLPs that obtains of the present invention, and diameter is that the VLPs of 30 nm and 55 nm diameters all is detected, and has and the similar form of natural dengue virus particle.
Fig. 9 is the immuno-electron microscope photo of the VLPs that obtains of the present invention; Wherein A, B are the VLPs in the cell pyrolysis liquid; C, D are the VLPs that expresses in the supernatant liquid concentrator, and used antibody is the dengue virus polyvalent antibody, also observes the VLPs that diameter is 30 nm and 55 nm diameters.
Figure 10 is the transmission electron microscope photo of the VLPs that obtains of the present invention; Wherein A is yeast X33 contrast; B is the transformant that the present invention makes up; In the endochylema of yeast cell, formed many duplicature vesica structures, all be full of diameter in each vesica and be about the VLPs that is the crystalloid arrangement about 30-55 nm.
Figure 11 is the time dependent level determination of the serum titer of VLPs immune mouse of the present invention; Wherein PBS is a PBS immunity Balb/C mouse negative control; PrME represents pGAPZ-sPrME VLPs immunity Balb/C mouse, and CPrME represents pGAPZ-sCPrME VLPs immunity Balb/C mouse.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
Confirming of the best construction strategy of embodiment 1 DENV1～4VLPs gene expression element
One, the extracting of DENV1～4 virus culture and viral RNA
DENV-1 GZ01/95 strain, DENV-2 ZS01/01 strain, DENV-3 H87 strain and DENV-4 H241 strain are inoculated respectively on the C6/36 cell monolayer; Add the MEM nutrient solution that contains 2% deactivation calf serum; Be cultured in 37 ℃ and cytopathy occur and reach " ++ +～++ ++ ", collect supernatant and carry out the preparation of RNA.Adopt Trizol reagent method to extract the total RNA of culture supernatant.Trizol
LS reagent is American I nvitrogen Company products.
Two, the preparation of DENV1～4 prME gene elements
Adopt ThermoScript
TMRT-PCR System (American I nvitrogen) is with synthetic cDNA first chain of the RNA reverse transcription of DENV1-4.
Reaction system: template ribonucleic acid (5-10 μ g) 8 μ l, primer R 2 μ l, dNTPs (100mM) 2 μ l, DEPC water 3 μ l.TV 15 μ l are with the mentioned component mixing and at 65 ℃ of water-bath 5 min, rapidly more than 2 min of precooling on ice.Add following ingredients: 5 * PrimeScript buffer, 4 μ l, RNase Inhibitor (40 U/ μ l) 1 μ l, Reverse Transcriptase (200 U/ μ l) 0.5 μ l.
Reaction conditions: 42 ℃ of insulation 30 min, 70 ℃ of 15 min.
The cDNA that obtains is used for pcr amplification, the PCR reaction system: 10 * LA Buffer (Mg
2+Free) 5 μ l, MgCl
25 μ l, dNTPs 8 μ l, cDNA 5 μ l, primers F, each 2 μ l of R, LA Taq enzyme 1 μ l, ddH
2O 22 μ l.
The structure and the evaluation of the recombinant vectors of embodiment 2 ability secreting, expressing DENV1～4VLPs
One, the structure of recombinant vectors
The prME gene of preparation among the embodiment 1 is reclaimed the purpose fragment behind Bsp119 I and Xba I double digestion, be connected, connect product and transform with the pGAPZ α A carrier (Invitrogen Company products) of the same double digestion of warp is directed
E.coliDH5 α (U.S. Stratagene Company products) competent cell, picking mono-clonal inoculation contain antibiotic LB substratum to carry out 37 ℃ of shaking culture and spends the night, the evaluation of cutting and check order of plasmid enzyme after preparation in a small amount.Choose and identify correct plasmid called after respectively: pGAPZ-α-PrMED1, pGAPZ-sPrME472-D2, pGAPZ-sPrME-D3 and pGAPZ-sPrME-D4; With identifying that correct recombinant vectors is transformed in the P.pastoris yeast cell; Obtain the engineering bacteria of stably express; Respectively at being preserved in Wuhan University China typical culture collection center on March 14th, 2011, numbering is respectively CCTCC M 2011063, CCTCC M 2011064, CCTCC M 2011065 and CCTCC M 2011066.
The structure synoptic diagram of above-mentioned recombinant vectors is as shown in Figure 1.
Two, recombinant vectors transforms pichia spp
With recombinant vectors [pGAPZ-α-PrMED1, pGAPZ-sPrME472-D2, pGAPZ-sPrME-D3, pGAPZ-sPrME-D4 ,] and the centrifugal back of the DH5 α culture bacteria liquid of empty carrier pGAP α A collect thalline, extract plasmid.With Bgl II enzyme respectively with above-mentioned recombinant plasmid linearizing.Reaction system: 10 * buffer L, 100 μ l, recombinant plasmid 200 μ l, Bgl II 50 μ l, ddH
2O 50 μ l.Behind the centrifugal mixing, be distributed into 20 μ l/ pipe, 37 ℃ of enzymes are cut and are spent the night.Get the pichia spp X33 competent cell that 100 μ l prepare, mix with the linearizing DNA of 10 μ g, the electricity of transferring to 0.2 cm of precooling transforms in the cup; Ice bath 5 min; The conversion of in Bio-Rad electricity conversion instrument, shocking by electricity, electric pulse parameter is voltage 1500 V, 5 ms.Add 1 mol/L sorbyl alcohol of 1 ml precooling after the electric shock immediately, mixed solution is transferred to 1.5 ml centrifuge tubes, 30 ℃ of incubation 1 h.Get 200 μ l and be layered on the YPD flat board that contains 100～500 μ g/ml Zeocin, 30 ℃ of constant temperature culture are cultivated and were grown oyster white yeast conversion bacterium colony to flat board in 2～3 days.
Three, the PCR of yeast transformant identifies
The above-mentioned yeast conversion bacterium colony of picking is with upstream and downstream universal primer 5 ' pGAPForward/3 ' the AOX I of Yeast expression carrier, α-factor/3 ' AOX I; Y1/3 ' AOX I carries out PCR respectively and detects PCR reaction system: 10 * Taq damping fluid, 5 μ l, dNTPs (2.5mM) 5 μ l; Yeast transformant 0.5 μ l; Each 2.5 μ l of upstream and downstream primer, rTaq enzyme (5U/ul) 1 μ l, ddH
2O 33.5 μ l.
The result is as shown in Figure 2.
Four, the Western blot of DENV1～4VLPs identifies
Picking is accredited as male through PCR and contains recombinant vectors [pGAPZ-α-PrMED1; PGAPZ-sPrME472-D2; PGAPZ-sPrME-D3, pGAPZ-sPrME-D4 ,] yeast transformant be inoculated in respectively in the 3 ml YPD liquid nutrient mediums (100 μ g/ml Zeocin); 30 ℃, 250 rpm aerobic shaking culture, 12 h.Then bacterium liquid is gone in the fresh YPD fermentation culture of 100 ml in the 1:100 ratio, 30 ℃ of shaking culture 120 h, 4 ℃ of 10,000 rpm is centrifugal, collect to go up cleer and peaceful thalline respectively, be stored in-70 ℃ subsequent use.With lysis buffer (50 mM NaH
2PO
4, 1 mM PMSF, 1 mM EDTA, 5% glycerol, pH 7.4) lysing cell.X33 transformant with empty carrier pGAP α A compares.
Get 80 μ l supernatants and mix with 20 μ l, 5 * sample-loading buffer, 20 μ l cell pyrolysis liquids mix with 2 * sample-loading buffer of equivalent, boil 10 min.After the sample cooling, carry out SDS-PAGE.Each point sample hole adds 30 μ l samples.Electrophoresis carries out coomassie brilliant blue staining with a glue after finishing, and another piece glue of identical point sample order carries out Western blotting and detects.Through half-dried electric commentaries on classics method; Albumen is transferred on the PDVF film, is one anti-with the anti-E monoclonal antibody (commercialization reagent) of DENV1～4 or with the polyvalent antibody of anti-DENV1～4 immunity, and HRP mark sheep anti-mouse igg is two anti-(1:2000 U.S. Sigma); The DAB colour developing, the result is as shown in Figure 6.All can detect E albumen and the 20 kDa prM albumen of about 50 kDa at the cell pyrolysis liquid of each recon and fermentation supernatant.
The preparation of embodiment 3 DENV1～4VLPs and evaluation
One, SDGC isolated viral appearance particle
The yeast cell that results are cultivated carries out purifying with 10～50% SDGU methods to DENV1-4 VLPs with the cell pyrolysis liquid supernatant, and absorption 30～40% contains cotton-shaped virus-like particle layer and carries out SDS-P Α GE and Western blotting detection.Fig. 7 is that the Western blot of purifying VLPs identifies figure.
Two, electron microscopy detects virus-like particle
The solution layer that contains virus-like particle that above-mentioned method of purification is obtained carries out the negative staining electron microscopic observation, and is as shown in Figure 8, and diameter is that the VLPs of 30 nm and 55 nm diameters all is detected.Anti-DENV-2 serum with certain weaker concn carries out immune electron microscopy, like Fig. 9, also observes the VLPs that diameter is 30 nm and 55 nm diameters.Collect the recombinant yeast cell of cultivating 72h and carry out the recombinant yeast cell ultrastructure that transmission electron microscope observing is expressed VLPs; Shown in figure 10; In the endochylema of yeast cell, formed many duplicature vesica structures, all be full of diameter in each vesica and be about the DENV-2 VLPs that is the crystalloid arrangement about 30-55 nm, no matter VLPs of the present invention is at morphological structure; Albumen and film component, even the DENV virus particle that in particulate assembling, all discharges with cells infected has similar characteristic.
The preliminary immunology research of embodiment 4 DENV1～4VLPs
One, immunization protocol
4-6 Balb/c mouse in age in week was injected DENV-1 VLPs or DENV-2 VLPs at 0,7,28 day, and inactivation of viruses immune group and PBS immune group are as contrast.
Two, ELISA detects serum antibody
1. the titration of standard antigen: encapsulate the VLPs of purifying, carry out doubling dilution.One anti-anti-VLPs serum for the 1:100 dilution.Measure OD
450nmAbsorption value, be the high dilution of antigen with the negative OD of positive OD/ greater than 2.1.
2. antigen coated: with the VLPs antigen of purifying, add in 96 orifice plates by 100 μ l/ holes, 37 ℃, 1h places 4 ℃ to spend the night then.PBST washes plate 3 times;
3. sealing: add the PBST of 4%BSA, 200 μ l/ holes, 37 ℃ of sealing 1h;
4. add the serum that begins to carry out each immune group of doubling dilution from 1:20,100 μ l/holes, 37 ℃ of incubation 1h, PBST wash plate 3 times;
5. add the HRP anti-mouse IgG of labelled goat (1:3000), 100 μ l/ holes, 37 ℃ of incubation 1h, PBST wash plate 3 times;
6. add tmb substrate 100 μ l/ holes, 37 ℃ of incubation 40 min.Add stop buffer 50 μ l/ holes, measuring wavelength is the OD value of 450 nm, is the highest antibody dilution multiple with positive serum OD/ negative serum OD greater than 2.1 serum dilution.The result is shown in figure 11.
Three, neutralization test detects the neutralization activity of serum antibody
1. DENV-2 NGC strain virus is made 10 times of serial dilutions (keeping liquid with MEM), the inoculating cell plate, every extent of dilution is inoculated 4 holes, and the pathology situation is observed in 37 ℃ of cultivations day by day, and record.Calculate TCID according to making 50% cell produce pathology at last
50
2. with the membrane filtration of each immune group serum with 0.22 μ m, then 56 ℃ of water-baths, deactivation 30 min.
3. the MEM with serum-free carries out doubling dilution 1:8 ~ 1:256 with serum.
4. keep liquid with MEM DENV-2 is diluted to 100 TCID
50
5. with 100 TCID of equivalent
50The DENV-2 diluent mixes with different dilution serum, then 37 ℃ of water-baths, in and 1h.Every extent of dilution kind 4 porocytes are at 37 ℃ of CO
2Incubator is cultivated observation of cell pathology and record after 7 days.If deactivation DENV-2 immune serum adds virus control, PBS serum adds virus control, and PBS serum adds the MEM contrast.
6. calculate in 50% serum and terminal point according to cytopathy, can protect 50% cell not produce the serum dilution of pathology.Be wherein and tire.The neutralization of surveying is tired and is 1:16～1:45.
Further, the present invention has also carried out Preliminary detection to the immunogenicity of DENV-3 VLPs and DENV-4 VLPs, and the result shows that the two equal can generation has the active antibody of certain neutralization.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (4)
1. dengue virus 1-4 virus-like particle recombinant vectors transforms
P .pastorisYeast strain; It is respectively pGAPZ-α-PrMED1-X33, pGAPZ-sPrME472-D2-X33, pGAPZ-sPrME-D3-X33 and pGAPZ-sPrME-D4-X33; Respectively at being preserved in Wuhan University China typical culture collection center on March 14th, 2011, numbering is respectively CCTCC M 2011063, CCTCC M 2011064, CCTCC M 2011065 and CCTCC M 2011066.
2. dengue virus 1-4 virus-like particle is characterized in that by the said recombinant vectors transfection of claim 1
P.pastorisYeast X33 cell justacrine gained.
3. the preparation method of dengue virus 1-4 virus-like particle is characterized in that comprising the steps:
1) adopt the RT-PCR technology from DENV-1 GZ01/95 strain, DENV-2 ZS01/01 strain, DENV-3 H87 strain and DENV-4 H241 strain, to obtain the prM-E gene element of four type dengue viruss respectively;
2) 1～4 type dengue virus CprM-E or prME expression of gene element are transformed,, replace the portion gene of four type dengue virus E gene anchorage zones at the signal peptide sequence of prM gene N end design ideal;
3) the prM-E genetic modification element with four type dengue viruss is cloned into respectively in the eukaryotic system expression vector;
4) with 3) in the transfection respectively of the recombinant expression vector that obtains
P.pastorisYeast X33 cell, and make its secreting, expressing dengue virus virus-like particle;
5) with SDS-PAGE and the genetic expression of Western blot testing goal;
6), detect the morphological specificity of virus-like particle with 15-60 quality % SDGC and electron microscopy.
4. dengue virus 1-4 virus-like particle is stepped on the application in the vaccine of removing from office type disease in the preparation prevention.
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