CN101948850A - Preparation method and application of virus-like particles of dengue viruses - Google Patents
Preparation method and application of virus-like particles of dengue viruses Download PDFInfo
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Abstract
The invention relates to a preparation method and application of virus-like particles (VLPs) of dengue viruses. The preparation method comprises the steps of: respectively modifying four types of dengue viruses (DENV) prM-E gene elements by means of a fusing PCR (Polymerase Chain Reaction) technology, then respectively cloning the four types of modified dengue viruses prM-E gene elements into a eukaryon system expression vector, respectively transfecting a mammalian cell with the recombinant expression vectors, and secreting the mammalian cells to express the virus-like particles of the dengue viruses. In addition, the invention lays a foundation for the development of a dengue viruses VLPs polyvaccine by carrying out preliminary study on the immune effects of DENV-1 VLPs and DENV-2 VLPs.
Description
Technical field
The present invention relates to the genetically engineered field, specifically, relate to the preparation method and the application of dengue virus virus-like particle.
Background technology
It is the widest popular arbovirus in the mankind nowadays that dengue virus (DENV) infects.Dengue virus is propagated by yellow-fever mosquito, nowadays almost in all torrid zones, subtropical zone distribution is arranged all.Estimate that according to WHO the whole world has 25～3,000,000,000 populations to face the danger of infecting dengue virus, annual nearly 5,000 ten thousand to 100,000,000 singapore hemorrhagic fever (Dengue fever DF) case takes place, and the patient shows symptoms such as high heat, headache, myalgia, arthrodynia and fash; This wherein has 500,000 examples to develop into even more serious dengue hemorrhagic fever (Dengue hemorrhagic fever DHF) and dengue shock syndrome (Dengue shock syndrome DSS), and annual number because of dengue virus infection death is 2.5 ten thousand.In recent years along with the development of global warming and tourist industry, the geographic range of its communication media yellow-fever mosquito constantly enlarges, dengue virus infection is extensively sent out in worldwide, has become one of the widest, that number of the infected maximum, harm the is maximum important arbovirus that distributes in the world.
But, also have many problems of not understanding so far and solving about dengue virus and infection thereof, so that the control of dengue virus infection lacks very effective measure, particularly dengue virus vaccine research does not make substantial progress.Dengue virus has 4 serotypes, and 4 kinds of serotype viruses can cause morbidity.Present studies show that; antibody at a certain serotype of dengue virus does not have long-term provide protection to other serotypes; when infecting special-shaped virus once more; (antibody-dependent enhancement ADE) makes the patient produce even more serious dengue hemorrhagic fever and dengue shock syndrome because antibody relies on enhancement in meeting.So a kind of successful stepping on removed from office vaccine and must can both be produced protective immunity to 4 type dengue viruss, can prevent the caused disease of dengue virus infection.WHO has been asserted dengue virus vaccine one of vaccine of first developing.And dengue virus vaccine also is the focus that the various countries scholar pays close attention to always, and nearly over 60 years, the effort that the leather vaccine is stepped in development never stops, and is used for clinical but still do not step on the leather vaccine at present.
Be accompanied by molecular biological develop rapidly, produced the new approaches that the leather vaccine is stepped in some researchs, virus sample particle vaccines is exactly wherein a kind of.Virus-like particle (VLPs) is a kind of pseudovirion that does not contain viral nucleic acid, there is not infectious composition fully, the true conformation that just has inactivation of viruses or attenuated live vaccine virion capsid, it presents virus antigen with the form of more approaching true conformation, discerned easilier, and do not have virus virulence to reply or the possibility of virogene reorganization or reprovision by body immune system.Just have very strong immunogenicity and good security, thereby be considered to an extraordinary vaccine candidate object owing to it.
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 in proper order '-NCR-C-PrM/M-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-NCR-3 ', 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 important biomolecules such as producing neutralizing antibody to learn function, 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 that for the Flavivirus member, co expression prM albumen and E albumen can form virus-like particle.
Because prM albumen and E albumen are glycoprotein, and the E.coli expression system itself has limitation, promptly lack rhetorical function behind the protein, make that expression product can not glycosylation.Therefore, in eukaryotic expression system, express the realization that prM albumen and E albumen help protein function.At present, still find no the report of secreting, expressing four type dengue virus virus-like particles research in eukaryotic system in the domestic research.
Summary of the invention
The preparation method who the purpose of this invention is to provide the dengue virus virus-like particle.
But another object of the present invention provides the eukaryotic system recombinant vectors of secreting, expressing DENV1-4 virus-like particle.
Further purpose of the present invention provides the application of dengue virus virus-like particle in the caused disease of prevention dengue virus.
In order to realize the object of the invention, the gene of coding dengue virus virus-like particle of the present invention, its have the nucleotide sequence shown in Seq ID No.1, Seq ID No.2, Seq ID No.3, Seq ID No.4 or the SeqID No.5 or this sequence through replace, lack or add that one or several Nucleotide forms have a proteinic nucleotide sequence of coding same function.The section of coding JESS signal peptide sequence in SeqIDNo.1-5 for example replaces with CCACAGGCACAGGCC with the GCTTGTGCAGGAGCC of coding 20-24 amino acids sequence.
The present invention also provides the carrier of the gene that contains coding dengue virus virus-like particle.Preferably, described carrier is the eukaryotic system expression vector.More preferably, described carrier is pcDNA5/FRT.
The present invention also provides the host cell that contains above-mentioned recombinant vectors.Preferably, described host cell is a mammalian host cell.More preferably, described host cell is the 293T cell.
The present invention also provides the preparation method of the dengue virus virus-like particle of said gene coding, and it comprises step:
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 dengue virus prM-E gene element of four types respectively; 2) respectively the dengue virus prM-E gene element of four types is transformed by merging round pcr, promptly before each type prM-E gene, increased by one section from the signal peptide sequence of japanese encephalitis virus SA14-14-2 strain and/or 20% gene order of each type prM-E gene C end is replaced with the corresponding sequence of japanese encephalitis virus SA14-14-2 strain E gene; 3) the dengue virus prM-E genetic modification element with four types is cloned into respectively in the eukaryotic system expression vector; 4) with 3) in the recombinant expression vector that obtains transfection mammalian cell respectively, and make its secreting, expressing dengue virus virus-like particle; 5) ELISA detects the immunogenicity of dengue virus virus-like particle.
The present invention further provides the application of dengue virus virus-like particle in the caused disease of prevention dengue virus of said gene coding.
Particularly, one aspect of the present invention provides the prM-E gene element of four type dengue viruss transforming through the design of genetic characteristics analysis and secreting, expressing.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-4H241 strain.In order to realize the secreting, expressing of dengue virus VLPs, the present invention carries out the secreting, expressing design to the prM-E gene element of four type dengue viruss and by merging round pcr the prM-E gene is transformed.Consider that in the mammalian cell expression system key that forms dengue virus VLPs is that correct synthetic, transhipment, cutting, packing and the conformation of prM-E albumen in express cell forms, the signal peptide sequence of prM gene N end has a direct impact it, and 20% zone of E gene C end has been played crucial effects for striding film district and helical region for the formation of dengue virus VLPs.Therefore the transformation of above-mentioned Expression element increases by one section signal peptide sequence from japanese encephalitis virus (JEV) SA14-14-2 strain before being included in DENV1-4 prM-E gene, and the corresponding sequence that 20% gene order of DENV1-4E gene C end is lacked or replaces with JEV SA14-14-2 strain E gene.
Improved four type dengue virus prM-E gene elements are called after respectively:
JD1prME、JD1prMEΔ20％、JD1prMEΔ20％JEV；
JD2prME、JD2prMEΔ20％、JD2prMEΔ20％JEV；
JD3prME、JD3prMEΔ20％、JD3prMEΔ20％JEV；
JD4prME、JD4prMEΔ20％、JD4prMEΔ20％JEV。
Another aspect of the present invention, provide can four type dengue viruss of secreting, expressing virus-like particles recombinant vectors.It is by special restriction enzyme site NheI and NotI, respectively four above-mentioned type dengue virus prM-E genetic modification elements is cloned among the eukaryotic system expression vector pcDNA5/FRT.The difference called after:
pJD1prME、pJD1prMEΔ20％、pJD1prMEΔ20％JEV；
pJD2prME、pJD2prMEΔ20％、pJD2prMEΔ20％JEV；
pJD3prME、pJD3prMEΔ20％、pJD3prMEΔ20％JEV；
pJD4prME、pJD4prMEΔ20％、pJD4prMEΔ20％JEV。
Further, the present invention gathers in the crops above-mentioned recombinant expression vector transfection 293T cell transfectional cell and expresses supernatant after 48 hours.Detect through IFA, DENV1-4 prM-E gene all obtains to efficiently express in cell.Detect the expression supernatant by Western blot, wherein, pJD2-4prME does not detect obvious purpose band, pJD1-4prME Δ 20% detects protein excretion, but be incomplete prM-E protein excretion, and, observe the purpose band at about 60KD place for pJD1prME and pJD1-4prME Δ 20%JEV, confirm that DENV1-4VLPs all effectively is secreted in the supernatant.
According to The above results, the present invention with pJD1prME and pJD1-4prME Δ 20%JEV classify as can secreting, expressing dengue virus virus-like particle recombinant vectors.
Of the present invention more on the one hand, a kind of preparation method of dengue virus virus-like particle is provided.It is with five above-mentioned a large amount of transfection mammalian cells of recombinant expression vector, express supernatant after high power concentrates, adopt the 15-60% sucrose density gradient centrifugation that DENV1-4VLPs is carried out purifying, and utilize SDS-PAGE and Western blot that the VLPs of purifying is identified.
Further, the present invention uses DENV-1 VLPs and DENV-2 VLPs immunity Balb/c mouse respectively, can produce the proteic special IgG antibody of anti-DENV rEIII, and antibody has certain neutralization activity, for dengue virus vaccine development is laid a good foundation.
The present invention is secreting, expressing four type dengue virus virus-like particles in eukaryotic expression system first, and the immune effect of DENV-1 VLPs and DENV-2 VLPs is carried out preliminary study, for the development of dengue virus VLPs polyvalent vaccine is laid a good foundation.
Description of drawings
Fig. 1 a is the pJD1-4prME recombinant vectors structural representation that the present invention has Japanese encephalitis signal peptide and prM-E full length gene.
Fig. 1 b is the pJD1-4prME Δ 20% recombinant vectors structural representation that the present invention has Japanese encephalitis signal peptide and terminal 20% gene of disappearance E gene C.
Fig. 1 c is that the present invention has the Japanese encephalitis signal peptide and terminal 20% gene of E gene C replaced with the pJD1-4prME Δ 20%JEV recombinant vectors structural representation of Japanese encephalitis respective regions.
Fig. 2 a is the identified by immunofluorescence figure that the present invention contains the pJD1-4prME cell, after wherein A, B, C, D represent pJD1prME, pJD2prME, pJD3prME, pJD4prME transient transfection 293T cell respectively, with the reaction result of stepping on leather E protein specific antibody DE1.
Fig. 2 b is the identified by immunofluorescence figure that the present invention contains the cell of pJD1-4prME Δ 20%, after wherein A, B, C, D represent pJD1prME Δ 20%, pJD2prME Δ 20%, pJD3prME Δ 20%, pJD4prME Δ 20% transient transfection 293T cell respectively, with the reaction result of stepping on leather E protein specific antibody DE1.
Fig. 2 c is the identified by immunofluorescence figure that the present invention contains pJD1-4prME Δ 20%JEV cell, after wherein A, B, C, D represent pJD1prME Δ 20%JEV, pJD2prME Δ 20%JEV, pJD3prME Δ 20%JEV, pJD4prME Δ 20%JEV transient transfection 293T cell respectively, with the reaction result of stepping on leather E protein specific antibody DE1.
Fig. 3 a is that expression supernatant behind pJD1prME of the present invention, pJD1prME Δ 20%, the pJD1prME Δ 20%JEV transfection 293T cell is through concentrating back and the DV1 E III albumen rabbit immune serum reaction result's that recombinates western blot evaluation figure, wherein 1,2,3,4 represent pJD1prME, pJD1prME Δ 20%, pJD1prME Δ 20%JEV and pcDNA5/FRT transient transfection 293T cell after 48 hours respectively, results are expressed supernatant, concentrate about 20 times after with the reaction result of reorganization DV1 E III albumen rabbit immune serum.
Fig. 3 b is that expression supernatant behind pJD2prME of the present invention, pJD2prME Δ 20%, the pJD2prME Δ 20%JEV transfection 293T cell is through concentrating back and the DV2 E III albumen rabbit immune serum reaction result's that recombinates western blot evaluation figure, wherein 1,2,3,4 represent pJD2prME, pJD2prME Δ 20%, pJD2prME Δ 20%JEV and pcDNA5/FRT transient transfection 293T cell after 48 hours respectively, results are expressed supernatant, concentrate about 20 times after with the reaction result of reorganization DV2 E III albumen rabbit immune serum.
Fig. 3 c is that expression supernatant behind pJD3prME of the present invention, pJD3prME Δ 20%, the pJD3prME Δ 20%JEV transfection 293T cell is through concentrating back and the DV3 E III albumen rabbit immune serum reaction result's that recombinates western blot evaluation figure, wherein 1,2,3,4 represent pJD3prME, pJD3prME Δ 20%, pJD3prME Δ 20%JEV and pcDNA5/FRT transient transfection 293T cell after 48 hours respectively, results are expressed supernatant, concentrate about 20 times after with the reaction result of reorganization DV3 E III albumen rabbit immune serum.
Fig. 3 d is that expression supernatant behind pJD4prME of the present invention, pJD4prME Δ 20%, the pJD4prME Δ 20%JEV transfection 293T cell is through concentrating back and the DV4 E III albumen rabbit immune serum reaction result's that recombinates western blot evaluation figure, wherein 1,2,3,4 represent pJD4prME, pJD4prME Δ 20%, pJD4prME Δ 20%JEV and pcDNA5/FRT transient transfection 293T cell after 48 hours respectively, results are expressed supernatant, concentrate about 20 times after with the reaction result of reorganization DV4 E III albumen rabbit immune serum.
Fig. 4 is the SDS-PAGE evaluation figure of DENV-1 and DENV-2 VLPs behind the ultracentrifugation purifying of the present invention, wherein M is albumen marker, 1 for super from after the supernatant layer, 2 is 20% sucrose layer, 3 for pJD1prME super from the bright band layer, 4 is that pJD2prME Δ 20%JEV is super from the bright band layer, 5 is 60% sucrose layer, 6 for pcDNA5/FRT super from after the supernatant layer, 7 is the 20% sucrose layer of pcDNA5/FRT, 8 is the bright band layer of pcDNA5/FRT, and 9 is the 60% sucrose layer of pcDNA5/FRT.
Fig. 5 is the Western blot evaluation figure of DENV-1 and DENV-2 VLPs behind the ultracentrifugation purifying of the present invention, wherein 1 for super from after the supernatant layer, 2 is 20% sucrose layer, 3 for pJD1prME is super from the bright band layer, and 4 is that pJD2prME Δ 20%JEV is super from the bright band layer, and 5 is 60% sucrose layer, 6 for pcDNA5/FRT super from after the supernatant layer, 7 is the 20% sucrose layer of pcDNA5/FRT, and 8 is the bright band layer of pcDNA5/FRT, and 9 is the 60% sucrose layer of pcDNA5/FRT; Reaginic antibody is reorganization DV E III albumen rabbit immune serum.
After Fig. 6 is DENV-1 VLPs of the present invention, DENV-2 VLPs and PBS immunity Balb/C mouse, the anti-rEIII protein I of mice serum gG detected result relatively, PBS is a PBS immunity Balb/C mouse negative control, D1 VLPs represents DENV-1 VLPs immunity Balb/C mouse, and D2 VLPs represents DENV-2 VLPs immunity Balb/C mouse.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The transformation of embodiment 1 DENV1-4prME gene element
One, the cultivation of DENV1-4, JEV and cell total rna extract
DENV-1 GZ01/95 strain, DENV-2 ZS01/01 strain, DENV-3 H87 strain and DENV-4 H241 strain, JEV SA14-14-2 strain (Chengdu Inst. of Biological Products) seed culture of viruses liquid are inoculated in the adherent C6/36 cell that covers with respectively, treat that cytopathy reaches ++ +～++ ++ the time, adopt Trizol reagent method to extract cell total rna.Trizol
LS reagent is American I nvitrogen company product.
Two, the transformation of DENV1-4 prME gene element
Adopt ThermoScript
TMRT-PCR System (American I nvitrogen) is with synthetic cDNA first chain of the RNA reverse transcription of DENV 1-4 and JEV.By the method that merges PCR the prM-E gene of DENV1-4 is transformed: (1) first round PCR increases respectively from 20% gene of the signal peptide sequence JESS of japanese encephalitis virus SA14-14-2 strain, DENV1-4 prME, DENV1-4prME Δ 20% and JEV E gene C end; (2) merging PCR is template with first round PCR product, respectively amplification:
JD1prME、JD1prMEΔ20％、JD1prMEΔ20％JEV；
JD2prME、JD2prMEΔ20％、JD2prMEΔ20％JEV；
JD3prME、JD3prMEΔ20％、JD3prMEΔ20％JEV；
JD4prME、JD4prMEΔ20％、JD4prMEΔ20％JEV。
Restriction enzyme site is NheI (GCTAGC) and NotI (GCGGCCGC).
First round PCR the primer is as follows:
JESS-F：5′-gggcGCTAGCCGCCGCCGCCATGGGAAAACGGTCAGCGGGCTCAATCATGTGGC-3′
JESS-R：5′-GGCTCCTGCACAAGCTATG-3′
DENV-1?prME-F(DENV-1?prMEΔ20％-F)：5′-CATAGCTTGTGCAGGAGCCTTCCATCTGACCACCCGAGG-3′
DENV-1?prME-R：5′-tgtgGCGGCCGCttaCGCCTGGACCATGACTCCTAGG-3′
DENV-1?prMEΔ20％-R：5′-tgtgGCGGCCGCttaACTGCTTCCCTTCTTGAACC-3′
r?DENV-1?prMEΔ20％-R：5′-GTTGAAAAGGCCTTGCCCAGACTGCTTCCCTTCTTGAACC-3′
DENV-2prM?E-F(DENV-2?prMEΔ20％-F)：5′-CATAGCTTGTGCAGGAGCCTTCCATTTAACCACACGCAACG-3′
DENV-2?prME-R：5′-tgtgGCGGCCGCttaGGCCTGCACCATGACTCCC-3′
DENV-2?prMEΔ20％-R：5′-tgtgGCGGCCGCttaAGAGCTTCCTTTCTTAAACC-3′
r?DENV-2?prMEΔ20％-R：5′-GTTGAAAAGGCCTTGCCCAGAGAGCTTCCTTTCTTAAACC-3′
DENV-3prM?E-F(DENV-3?prMEΔ20％-F)：5′-CATAGCTTGTGCAGGAGCCTTCCACTTAACTTCACGAGATGG-3′
DENV-3?prME-R：5′-tgtgGCGGCCGCttaAGCTTGCACCACGACCCCCAG-3′
DENV-3?prMEΔ20％-R：5′-tgtgGCGGCCGCttaCGAGCTTCCCTTCCTGTACC-3′
r?DENV-3?prMEΔ20％-R：5′-GTTGAAAAGGCCTTGCCCAGCGAGCTTCCCTTCCTGTACC-3′
DENV-4?prM?E-F(DENV-4?prMEΔ20％-F)：5′-CATAGCTTGTGCAGGAGCCTTTCACTTGTCAACAAGAGATGG-3′
DENV-4?prME-R：5′-tgtgGCGGCCGCttaTGCGTGAACTGTGAAACCCAG-3′
DENV-4?prMEΔ20％-R：5′-tgtgGCGGCCGCttaGGAACTCCCTTTCCTGAACCAATGG-3′
r?DENV-4?prMEΔ20％-R：5′-GTTGAAAAGGCCTTGCCCAGGGAACTCCCTTTCCTGAACC-3′
JEV?E20％-F：5′-CTGGGCAAGGCCTTTTCAAC-3′
JEV?E20％-R：5′-tgtgGCGGCCGCttaAGCATGCACATTGGTCGCTAAG-3′
First round PCR system and condition (employing Luo Shi test kit: PCR Grade Nucleotide Mix)
1. 20% gene PCR system: ddH of JESS and JEV E gene C end increases respectively
2O 36 μ l; 10 * damping fluid, 5 μ l; DMSO 2.5 μ l; DNTP1 μ l; Primers F, each 2 μ l of R; Template 1 μ l; Enzyme 0.5 μ l.Condition: 95 ℃ of 2min
72℃?7min
2. DENV1-4 prME and the DENV1-4 prME Δ 20% gene PCR system of increasing respectively is the same.
Condition: 95 ℃ of 2min
72℃?7min
It is as follows to merge the PCR the primer:
DENV-1?prME-F(DENV-1?prMEΔ20％-F)：5′-CATAGCTTGTGCAGGAGCCTTCCATCTGACCACCCGAGG-3′
DENV-2prM?E-F(DENV-2?prMEΔ20％-F)：5′-CATAGCTTGTGCAGGAGCCTTCCATTTAACCACACGCAACG-3′
DENV-3prM?E-F(DENV-3?prMEΔ20％-F)：5′-CATAGCTTGTGCAGGAGCCTTCCACTTAACTTCACGAGATGG-3′
DENV-4?prM?E-F(DENV-4?prMEΔ20％-F)：5′-CATAGCTTGTGCAGGAGCCTTTCACTTGTCAACAAGAGATGG-3′
JEV?E20％-R：5′-tgtgGCGGCCGCttaAGCATGCACATTGGTCGCTAAG-3′
JESS-F：5′-gggcGCTAGCCGCCGCCGCCATGGGAAAACGGTCAGCGGGCTCAATCATGTGGC-3′
DENV-1?prME-R：5′-tgtgGCGGCCGCttaCGCCTGGACCATGACTCCTAGG-3′
DENV-1?prMEΔ20％-R：5′-tgtgGCGGCCGCttaACTGCTTCCCTTCTTGAACC-3′
DENV-2?prME-R：5′-tgtgGCGGCCGCttaGGCCTGCACCATGACTCCC-3′
DENV-2?prMEΔ20％-R：5′-tgtgGCGGCCGCttaAGAGCTTCCTTTCTTAAACC-3′
DENV-3?prME-R：5′-tgtgGCGGCCGCttaAGCTTGCACCACGACCCCCAG-3′
DENV-3?prMEΔ20％-R：5′-tgtgGCGGCCGCttaCGAGCTTCCCTTCCTGTACC-3′
DENV-4?prME-R：5′-tgtgGCGGCCGCttaTGCGTGAACTGTGAAACCCAG-3′
DENV-4?prMEΔ20％-R：5′-tgtgGCGGCCGCttaGGAACTCCCTTTCCTGAACCAATGG-3′
Merge PCR system and condition (employing Luo Shi test kit: PCR Grade Nucleotide Mix)
1. at first respectively 20% gene of DENV1-4 prME Δ 20% and JEV E gene C end is merged the fragment called after DENV1-4 prME Δ 20%JEV after the fusion.
PCR system: ddH
2O 35 μ l; 10 * damping fluid, 5 μ l; DMSO 2.5 μ l; DNTP1 μ l; Primers F, each 2 μ l of R; Two each 1 μ l of segmental template; Enzyme 0.5 μ l.
Condition: 95 ℃ of 2min
72℃?7min
2. at last JESS is merged with DENV1-4 prME, DENV1-4 prME Δ 20% and DENV1-4 prME Δ 20%JEV gene respectively.
PCR system: ddH
2O 35 μ l; 10 * damping fluid, 5 μ l; DMSO 2.5 μ l; DNTP1 μ l; Primers F, each 2 μ l of R; Two each 1 μ l of segmental template; Enzyme 0.5 μ l.
Condition: 95 ℃ of 2min
72℃?7min
To behind NheI and NotI double digestion, reclaim the purpose fragment through the prME gene that merges the PCR transformation among the embodiment 1, be connected with pcDNA5/FRT carrier (Invitrogen company product) orientation through same double digestion, connect product Transformed E .coli DH5 α (U.S. Stratagene company product) competent cell, picking mono-clonal inoculation contains antibiotic LB substratum and carried out 37 ℃ of shaking culture 12 hours, the evaluation of cutting and check order of plasmid enzyme after preparation in a small amount.Choose and identify correct plasmid called after respectively:
pJD1prME、pJD1prMEΔ20％、pJD1prMEΔ20％JEV；
pJD2prME、pJD2prMEΔ20％、pJD2prMEΔ20％JEV；
pJD3prME、pJD3prMEΔ20％、pJD3prMEΔ20％JEV；
pJD4prME、pJD4prMEΔ20％、pJD4prMEΔ20％JEV；
The structural representation of above-mentioned recombinant expression vector is shown in Fig. 1 a～Fig. 1 c.
Two, recombinant vectors transient transfection 293T cell
Transfection the day before yesterday reaches 293T cell (ATCC) in 6 orifice plates with the substratum that does not contain resistance, treats that cell grows to 90% individual layer and carries out transfection when full.With not containing FBS and two anti-substratum wash twice gently with cell, add the above-mentioned substratum of 2ml before the transfection.The preparation transfection mixture: at first add 50 μ l OPTI (not containing serum) in the EP pipe, then add the plasmid that 2 μ g treat transfection, mixing adds 5 μ l transfection reagent (FuGENE more gently
HD Transfection Reagent), mixing gently, room temperature leaves standstill 20min, and transfection reagent is splashed in the cell culture medium gently, rotates Tissue Culture Plate gently, with the liquid mixing.37 ℃ of CO
2Incubator detects after cultivating 48h, and transfection is provided with negative control simultaneously, is pcDNA5/FRT empty carrier transfectional cell.
Three, the indirect immunofluorescence of DENV1-4 VLPs is identified
With supernatant gently sucking-off temporarily be stored in 4 ℃.Cell is blown down the back to be washed 2 times with PBS.Cell is resuspended among the PBS, preparation antigen sheet, and cold acetone is 15min fixedly.With dengue virus E protein specific antibody DE1 (abcam) is one anti-(1: 100), and the sheep anti mouse serum of FITC mark is two anti-(1: 100; U.S. Sigma), fluorescent microscope is observed the antigen sheet down.The result can observe specific fluorescence under fluorescent microscope, the 293T cell of pcDNA5/FRT transfection not with the DE1 antibody response.The result as shown in Figure 2.
Four, the western-blot of DENV1-4 VLPs identifies
Get 40 μ l after supernatant concentrated 20 times and add 10 μ l, 5 * albumen sample-loading buffer, boil 3min, carry out the SDS-PAGE electrophoresis, through half-dried electric commentaries on classics method, albumen is transferred on the PDVF film, is one anti-(1: 200) with the rabbit anteserum of DENV1-4 reorganization E III protein immunization, and the goat-anti rabbit anteserum of HRP mark is two anti-(1: 2000 U.S. Sigma), the DAB colour developing, the result as shown in Figure 3.PJD1prME and pJD1prME Δ 20%JEV be all visible purpose band at the 60KD place, and pJD1prME Δ 20% stripe size is significantly less than the above two.PJD2-4prME expresses supernatant and there is no obvious purpose band, and pJD2-4prME Δ 20%JEV expresses supernatant in the equal bands visible in 60KD place, and is consistent with expection, though and pJD2-4prME Δ 20% expression supernatant can detect the purpose band, size is significantly less than 60KD.
Preparation, evaluation and the immunogenicity experiments of embodiment 3 DENV1-4VLPs
One, the preparation of DENV1-4VLPs and evaluation
Results pJD1prME, each 2L of pJD1-4prME Δ 20%JEV transfection supernatant, high power adopts the 15-60% sucrose density gradient centrifugation that DENV1-4 VLPs is carried out purifying after being concentrated into 3ml, extracts each layer band and carries out SDS-PAGE and Western blot detection.Fig. 4 is that the SDS-PAGE of DENV-1 and DENV-2VLPs identifies figure, and Fig. 5 is that the Western blot of DENV-1 and DENV-2VLPs identifies figure.
Two, DENV-1 and the preliminary immunogenicity experiments of DENV-2 VLPs
1, 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 1 * PBS is as negative control.
2, (rEIII albumen is the EIII albumen of four type dengue viruss of tandem expression in prokaryotic expression system to the anti-rEIII protein antibodies of ELISA detection serum, its preparation process is as follows: at first, the EIII gene fragment of pcr amplification 1-4 type DENV, and by obtaining fusion gene rEIII after the EIII gene fragment connection of method with 1-4 type DENV of merging PCR, the rEIII fusion gene cloning to prokaryotic expression carrier pET30a, is expressed in e. coli bl21 (DE3).SDS-PAGE soluble analysis result shows that expression-form is an inclusion body.Renaturing inclusion bodies adopts the method for dilution refolding, and refolded protein carries out purifying through metal chelate chromatography post and weak anionic exchange column successively, promptly).
A. antigen coated: the rE III albumen with the prokaryotic expression purifying is antigen, adds in 96 orifice plates by the 200ng/ hole, and 4 ℃ are spent the night, and PBST washes plate 3 times;
B. every hole adds 5% skimmed milk of the aseptic PBS preparation of 300 μ l, 37 ℃ of sealing 2h, and PBST washes plate 3 times;
C. the mice serum collected is diluted with 5% skimmed milk, since 1: 50, make 2 times of serial dilutions, add in 96 orifice plates by 100 μ l/ holes, hatch 1h for 37 ℃, PBST washes plate 6 times;
D. adding two resists: by dilution in 1: 1000,1h was hatched for 37 ℃ in 100 μ l/ holes to the sheep anti-mouse igg of HRP mark with 5% skimmed milk, and PBST washes plate 6 times;
E. colour developing: add substrate A liquid, the every hole 50 μ l of B liquid (available from ten thousand safe biological medicine companies), lucifuge is placed 10min.2N H
2SO
450 μ l termination reactions.
F. microplate reader detects A
450, the result as shown in Figure 6.After Fig. 6 is DENV-1 VLPs, DENV-2VLPs and PBS immunity Balb/C mouse, the anti-rEIII protein I of mouse serum gG detected result relatively, wherein PBS is a PBS immunity Balb/C mouse negative control, D1VLPs represents DENV-1 VLPs immunity Balb/C mouse, and D2 VLPs represents DENV-2 VLPs immunity Balb/C mouse.
3, neutralization test detects the neutralization activity of serum
A. with BHK-21 passage to 96 orifice plate, treat that cell grows to individual layer;
B. with the mouse immune serum collected in 56 ℃ of deactivation 30min, cross 0.45 μ m filter membrane;
C. inactivated serum Eagle ' s is kept liquid and (contain 1%FBS, 1%P.S, 1%G, 2%Na
+) since 1: 5, make 2 times doubling dilution successively, mixed 37 ℃ of incubation 1h by 1: 1 with DENV-1 or the DENV-2 of 100TCID50;
D. the nutrient solution in sucking-off 96 orifice plates is washed 1 time with keeping liquid;
E. press in the adding of 200 μ l/ holes and serum, each extent of dilution is done 4 multiple holes;
F.37 ℃, 5%CO
2Condition is cultivated, and observes pathology day by day, observes altogether 7 days;
G. with four multiple Kong Jun do not occur observable cytopathic serum dilution as antibody neutralization tire, the result is as shown in table 1.
Table 1DENV-1 and DENV-2 VLPs mouse immune serum NAT detected result
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 above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
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| CN102363751A (en) * | 2011-03-24 | 2012-02-29 | 中山大学 | Dengue virus-like particle and its preparation method and application |
| CN105246491A (en) * | 2013-03-15 | 2016-01-13 | 宾夕法尼亚大学理事会 | Novel vaccine against multiple dengue virus subtypes |
| CN106687590A (en) * | 2014-09-11 | 2017-05-17 | Vlp 治疗有限责任公司 | yellow fever virus virus-like particle |
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| 《中华实验和临床病毒学杂志》 20091230 苗芳等 登革Ⅰ型病毒E蛋白在293T细胞中的分泌表达 第23卷, 第6期 2 * |
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| CN102363751A (en) * | 2011-03-24 | 2012-02-29 | 中山大学 | Dengue virus-like particle and its preparation method and application |
| CN105246491A (en) * | 2013-03-15 | 2016-01-13 | 宾夕法尼亚大学理事会 | Novel vaccine against multiple dengue virus subtypes |
| CN110055265A (en) * | 2013-03-15 | 2019-07-26 | 宾夕法尼亚大学理事会 | For the novel vaccine of a variety of subtypes of dengue virus |
| CN106687590A (en) * | 2014-09-11 | 2017-05-17 | Vlp 治疗有限责任公司 | yellow fever virus virus-like particle |
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