CN103205460B - A kind of embedded virus and its preparation method and application - Google Patents

Abstract

The invention discloses a kind of infection and encephalitis B virus infection sex clone, it is the recombinant vectors comprising Japanese encephalitis attenuated virus strain SA14-14-2 Genomic full_length cDNA; The invention also discloses a kind of embedded virus, and the purposes of this embedded virus; And a kind of SEQ? ID? bacterial artificial chromosome shown in NO.1.Embedded virus of the present invention can on various human production of vaccine cell growth and breeding, and growth characteristics are similar, may be used for the production of four serotype dengue vaccine, further, immunogenicity is strong, and the antibody titers of generation is high, have a good application prospect, have stronger industrial application value.

Description

A kind of embedded virus and its preparation method and application

Technical field

The present invention relates to a kind of embedded virus, particularly a kind of infection and encephalitis B virus infection sex clone being fitted together to dengue virus protective antigen.

Background technology

Singapore hemorrhagic fever is likened to one of harm unheeded Perenniporia martius transmissible disease the most serious by WHO.According to WHO statistics, since nineteen fifty-five finds this disease, the Dengue case of WHO report increases about 30 times.At present, the whole world has nearly half population (3,500,000,000) to live in the epidemic regions of singapore hemorrhagic fever, and annual morbidity example is approximately 5,000 ten thousand, but the whole world does not also have vaccine available at present.

The key difficulties of dengue vaccine research and development is that dengue virus is divided into four kinds by serotype, i.e. Dengue 1 type, Dengue 2 type, Dengue 3 type, Dengue 4 type, and alternately occurs in epidemic regions.Although have cross reaction between four serotypes, do not have cross protection or cross protection very weak, the time length is very short, only has the several months.When after the dengue virus infecting certain serotype, the general clinical symptom or asymptomatic that only reveal any symptoms is more weak, as again by another kind of serotype dengue virus infection, there will be the pathology enhancement of antibody-dependant, show as latent period short, clinical symptom obviously increases the weight of, and may occur serious hemorrhage, shock or dead.Therefore the tetravalence dengue vaccine simultaneously protecting 4 serotype dengue viruss must be researched and developed.Because 4 serotype dengue virus growth characteristics difference are large, immunne response ability is different, the balance that maintain immune response level certain between 4 type dengue vaccine has certain technical difficulty.Therefore, safe and effective vaccine is not had to go on the market so far.

Application number: 200910085231.6, denomination of invention: the patent application of Japanese encephalitis/dengue chimeric virus and application thereof utilizes encephalitis attenuated vaccine strain SA14-14-2 to be gene framework, replaces Vaccinum Encephalitidis Epidemicae strain corresponding gene region structure and forms by dengue 2-type virus NGC strain antigen gene prM-E.Because the patented product only can be bred on mosquito cells C6/36, this cell can not be used for the production of vaccine for man, and is only a kind of embedded virus of encephalitis/Dengue 2 type, cannot carry out production of vaccine, there is very large defect.

Summary of the invention

In order to solve the problem, the invention provides a kind of new infection and encephalitis B virus infection sex clone, the dengue chimeric virus being skeleton with this infection and encephalitis B virus infection sex clone, and dengue vaccine and new bacterial artificial chromosome.

Infection and encephalitis B virus infection sex clone of the present invention, it is the recombinant vectors comprising Japanese encephalitis attenuated virus strain SA14-14-2 Genomic full_length cDNA.

Wherein, described recombinant vectors is restructuring pACNR plasmid or recombinant bacteria artificial chromosome, and the nucleotide sequence of bacterial artificial chromosome is as shown in SEQIDNO.1.

Wherein, 5 ' end of described full-length cDNA has prokaryotic promoter.Described promotor is SP6.

The translational termination site that 3 ' of described full-length cDNA is held is restriction enzyme digestion sites.Described restriction enzyme digestion sites is XhoI or SalI.

Preferably, the nucleotide sequence of described infections clone is as shown in SEQIDNO.2 or SEQIDNO.3.

Embedded virus of the present invention, it is built by aforementioned infection and encephalitis B virus infection sex clone and forms, and wherein prM-E gene is dengue virus prM-E gene.

Wherein, described dengue virus is dengue virus 1,2,3 or 4 type.

Wherein, described dengue virus 1 type is dengue virus ThD0008-81 strain; Described dengue type 2 virus is dengue virus PUO-218 strain; Described dengue virus 3 type is dengue virus PaH881-88 strain; Described dengue virus 4 type is dengue virus 814669 strain.

Wherein, the nucleotide sequence of described dengue virus prM-E gene is respectively as shown in SEQIDNO.4 ~ 7.

The nucleotide sequence of described embedded virus is as shown in any one of SEQIDNO.8 ~ 11.

Aforementioned embedded virus is preparing the purposes in dengue vaccine.

Present invention also offers a kind of dengue vaccine, it is prepared from by aforesaid embedded virus.

The present invention is also supplied to the bacterial artificial chromosome of a kind of nucleotide sequence as shown in SEQIDNO.1.

The present invention utilizes encephalitis attenuated vaccine strain SA14-14-2 and bacterial artificial chromosome of the present invention, successfully construct infection and encephalitis B virus infection sex clone, and with this infections clone for gene framework, successfully construct containing Dengue 1 type, Dengue 2 type, the Japanese encephalitis/dengue chimeric virus of Dengue 3 type or 4-type dengue virus antigen gene prM-E, these embedded viruses can on various human production of vaccine cell growth and breeding, and growth characteristics are similar, may be used for the production of four serotype dengue vaccine, immunogenicity is strong, the titre of the antibody produced is high, have a good application prospect.

Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.

The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.

Accompanying drawing explanation

Figure 1B AC vector modification (BAC53) is Hind III & Kpn I double digestion proof diagram afterwards, 4 clones of swimming lane 1,2,3,4 for selecting, and M is DNA molecular amount standard DL15000);

The enzyme of Fig. 2 JEV5 ' half molecule plasmid pJEV5 ' structure cuts qualification result, and wherein the 1st swimming lane is DNAmarker (DL15000); 2nd swimming lane is plasmid vector pACNR; 3rd swimming lane F1 fragment is inserted into the pACNR-F1 after carrier pACNR to clone; 4th swimming lane is that F1 fragment and F3 fragment are inserted into the pACNR-F13 after carrier pACNR; 5th swimming lane is 5 ' half molecule plasmid pJEV5 ' F1, F2 and F3 fragment be inserted into after carrier pACNR; 6th swimming lane is the result of 5 ' half molecule plasmid pJEV5 ' restriction restriction endonuclease AscI and KasI being carried out to digestion verification, 7th swimming lane is the result of 5 ' half molecule plasmid pJEV5 ' restriction restriction endonuclease BglII and KasI being carried out to digestion verification, 8th swimming lane is the result of 5 ' half molecule plasmid pJEV5 ' restriction restriction endonuclease BglII and BspEI being carried out to digestion verification, 9th swimming lane is the result of 5 ' half molecule plasmid pJEV5 ' restriction restriction endonuclease AscI and Xhol being carried out to digestion verification, and the 10th swimming lane is DNAmarker (DL2000);

Fig. 3 pJFC total length plasmid construct schematic diagram, red arrow represents that people is the promoter sequence added, and AscI restriction enzyme site is for people is for adding for cloning structure, and blue portion represents encephalitis attenuated vaccine strain SA14-14-2 full-length cDNA;

Fig. 4 pJFC total length plasmid enzyme restriction qualification result, wherein swimming lane 1 is DNAmarkerDL15000; Swimming lane 2 is pJFC total length plasmid; Swimming lane 3 is the electrophoretogram that pJFC cuts through HindIII enzyme; Swimming lane 4 is the electrophoretogram that pJFC cuts through BglII enzyme; Swimming lane 5 is DNAmarkerDL2000;

Fig. 5 BAC53-JFC total length plasmid enzyme restriction qualification result, wherein swimming lane 1 is the electrophoretogram that BAC53-JFC cuts through NotI and XhoI enzyme; Swimming lane 2 is DNAmarkerDL2000; M is DNAmarkerDL15000;

Fig. 6 RT-PCR detects the electrophoresis result of rJEV.Swimming lane 1 is DNAmarker (λ DNA/HindIII), swimming lane 2 is DNAmarker (DL2000), swimming lane 3 is pcr amplification F123 fragment, swimming lane 4 is pcr amplification F45 fragment, swimming lane 5 is pcr amplification F67 fragment swimming lane 6 is pcr amplification F89 fragment, swimming lane 7 is pcr amplification F10 fragment, and swimming lane 8 is pcr amplification F11 fragment;

Fig. 7 indirect immunofluorescene assay rJEV result.Wherein A, B are by the result that JEVE monoclonal antibody and JEVNS1 monoclonal antibody detect respectively after attenuated strain JEV infection cell; C, D are by the result that JEVE monoclonal antibody and JEVNS1 monoclonal antibody detect respectively after recovered virus rJEV cells infected; E, F are the result of cell controls JEVE monoclonal antibody and the JEVNS1 monoclonal antibody detection do not infected respectively;

Fig. 8 rJEV compares with the plaque form of Vaccinum Encephalitidis Epidemicae strain JEV;

5 ' half molecular cloning pJD1-5 ' the plasmid electrophorogram that Fig. 9 encephalitis/Dengue 1 type is chimeric;

The electrophorogram of 5 ' half molecular cloning pJD1-5 ' the plasmid NotI that Figure 10 encephalitis/Dengue 1 type is chimeric and BglII double digestion;

The plasmid pJD1FC electrophorogram of Figure 11 encephalitis/Dengue 1 type chimaeric full length clone;

The plasmid pJD1FC NotI of Figure 12 encephalitis/Dengue 1 type chimaeric full length clone and the electrophorogram of XhoI double digestion;

The plasmid pJD1FC of Figure 13 encephalitis/Dengue 1 type chimaeric full length clone uses the electrophorogram of BamHI and XhoI single endonuclease digestion respectively;

The electrophorogram of the plasmid pJD1FC in-vitro transcription product of Figure 14 encephalitis/Dengue 1 type chimaeric full length clone;

Cytopathy after the plasmid pJD1FC in-vitro transcription product transfectional cell BHK21 of Figure 15 encephalitis/Dengue 1 type chimaeric full length clone, left side figure is the cell controls of untransfected, and the right is the cytopathy after transfection;

Pathology result after Figure 16 embedded virus JD1 goes down to posterity on primary hamster kidney cell, the left side is the cell controls do not infected, and the right is metainfective cytopathy;

The electrophorogram of the RT-PCR calibrating of Figure 17 embedded virus JD1, amplified fragments 1,2,3 is all consistent with expection;

The plaque detected result of Figure 18 encephalitis/Dengue 1 type embedded virus JD1;

In Figure 19 encephalitis/Dengue 1 type embedded virus JD1, dengue virus 1 type E protein expresses Westernblot detected result;

The growth characteristics detected result of Figure 20 encephalitis/Dengue 1 type embedded virus JD1 on primary hamster kidney cell PHK;

The immunogenicity detected result of Figure 21 encephalitis/Dengue 1 type embedded virus JD1;

Figure 22 encephalitis/Dengue 2 type chimaeric full length clone pJD2FC enzyme cuts qualification.1 is DNAmarker (DL15000); 2 is pJD2FC plasmid; 3 is pJD2FC plasmid HindIII restriction enzyme mapping; 4 is pJD2FC plasmid BglII restriction enzyme mapping; 5 is pJD2FC plasmid double digestion collection of illustrative plates (AscI+Xhol); 6 is pJD2FC plasmid double digestion collection of illustrative plates (BspEI+Xhol); 7 is DNAmarker (DL2000);

The cytopathy of Figure 23 encephalitis/Dengue 2 type embedded virus JD2 on primary hamster kidney cell, the left side is not for infect compared with control cells, and the right is the cytopathy after JD2 infects PHK cell;

Figure 24 RT-PCR detects the electrophorogram of embedded virus JD2.1 is DNAmarker (DL15000); 2 is pcr amplified fragment F123; 3 is pcr amplified fragment F45; 4 is pcr amplified fragment F67; 5 is pcr amplified fragment F89; 6 is pcr amplified fragment F10; 7 is pcr amplified fragment F11;

The experimental result of Figure 25 indirect immunofluorescene assay encephalitis/Dengue 2 type embedded virus JD2.Wherein A, B, C are respectively by the result that Dengue 2 type monoclonal antibody, JEVE monoclonal antibody and JEVNS1 monoclonal antibody detect after dengue 2-type virus cells infected; D, E, F are that embedded virus JD2 is respectively by the result that Dengue 2 type monoclonal antibody, JEVE monoclonal antibody and JEVNS1 monoclonal antibody detect; G, H, I are that encephalitis recovered virus rJEV is respectively by the result that Dengue 2 type monoclonal antibody, JEVE monoclonal antibody and JEVNS1 monoclonal antibody detect; J, K, L are that the cell of uninfecting virus is respectively by the result that E monoclonal antibody and the JEVNS1 monoclonal antibody of Dengue 2 type monoclonal antibody, JEV detect;

Figure 26 encephalitis/Dengue 2 type embedded virus JD2 plaque form and size;

The growth curve of Figure 27 encephalitis/Dengue 2 type embedded virus JD2 on primary hamster kidney cell;

The immunogenicity detected result of Figure 28 encephalitis/Dengue 2 type embedded virus JD2;

Figure 29 has chimeric 5 ' half molecule pJD3-5 ' double digestion (Kas I/Bgl II) the qualification electrophorogram of encephalitis/Dengue 3 type of 8 base deletions;

Figure 30 fusion DNA vaccine amplification 1-3446 fragment, eliminates 8 bases of disappearance;

Chimeric 5 ' half molecule BAC53JD3-5 ' the double digestion qualification electrophorogram of Figure 31 encephalitis/Dengue 3 type, swimming lane 1 is BamH I single endonuclease digestion, and swimming lane 2 is Asc I/BamH I double digestion;

Figure 32 is for building the JEV-F11 fragment PCR amplification electrophorogram of JEV3 ' the half molecule pJEV-3 ' (Sal I) containing SalI restriction enzyme site;

Figure 33 cuts qualification electrophorogram containing JEV3 ' half molecule pJEV3 ' (SalI) enzyme of SalI restriction enzyme site, and swimming lane 1 is Xho I/Xba I double digestion, and swimming lane 2 is SalI/XbaI double digestion;

Figure 34 encephalitis/Dengue 3 type chimaeric full length clone BAC53JD3FC plasmid HindIII limits restriction endonuclease and carries out the electrophorogram that enzyme cuts qualification, and swimming lane 1 is pJD3 plasmid, and M1 is molecular weight standard DL15,000, M2 is molecular weight standard DL2, and 000;

Figure 35 encephalitis/Dengue 3 type chimaeric full length clone BAC53JD3FC plasmid XhoI limits restriction endonuclease and carries out the electrophorogram that enzyme cuts qualification, and swimming lane 1 is pJD3 plasmid, and M is molecular weight standard DL15, and 000;

Figure 36 encephalitis/Dengue 3 type chimaeric full length clone BAC53-JD3FC in-vitro transcription RNA electrophorogram, 1 be in-vitro transcription RNA, M1 be molecular weight standard DL15,000, M2 is molecular weight standard DL2,000;

The pathology situation of Figure 37 PHK cell, compared with compared with control cells (left side), there is obvious CPE in the cell (right side) having infected encephalitis/Dengue 3 type embedded virus;

Figure 38 encephalitis/Dengue 3 type embedded virus adopts the RT-PCR qualification result of Dengue 1 ~ Dengue 4 type specific detection primer, only has Dengue 3 type to detect primer amplification and goes out the fragment that length is 615bp;

The plaque detected result of Figure 39 encephalitis/Dengue 3 type embedded virus is 5.93lgPFU/ml according to the virus titer that plaque counting obtains;

Figure 40 encephalitis/Dengue 3 type embedded virus growth curve is 0.001 according to result MOI is best infective dose;

The Westernblot qualification of Figure 41 encephalitis/Dengue 3 type embedded virus.Result display embedded virus can be observed clear band in dengue virus E protein corresponding position, and encephalitis b virus has no band;

The immunogenicity determinations of Figure 42 encephalitis/Dengue 3 type embedded virus JD3;

The electrophoretogram of Figure 43 encephalitis/Dengue 4 type 5 ' half molecular cloning pJD4-5 ' plasmid.PJD4-5 ' half plasmid size is 6.3kb;

The enzyme of Figure 44 encephalitis/Dengue 4 type 5 ' half molecular cloning pJD4-5 ' plasmid cuts qualification collection of illustrative plates.PJD4-5 ' plasmid is 4.1kb and 2.2kb fragment through KasI and BglII double digestion;

The electrophoretogram of Figure 45 encephalitis/Dengue 4 type full-length clone pJD4FC plasmid.PJD4FC plasmid size is 13.6kb;

The enzyme of Figure 46 encephalitis/Dengue 4 type full-length clone pJD4FC plasmid cuts qualification collection of illustrative plates.PJD4FC plasmid is 8.9kb and 4.7kb fragment through BspEI and MluI double digestion;

The electrophoretogram of Figure 47 pJD4FC in-vitro transcription product;

The pathology situation of Figure 48 BHK21 cell, compared with compared with control cells (left side), there is obvious CPE in the cell (right side) of transfection pJD4FC in-vitro transcription thing;

The pathology situation of Figure 49 PHK cell, compared with compared with control cells (left side), there is obvious CPE in the cell (right side) having infected encephalitis/Dengue 4 type embedded virus JD4;

The RT-PCR qualification result of Figure 50 encephalitis/Dengue 4 type embedded virus, adopts 4 pairs of primers to amplify the fragment that length is 2078bp, 2155bp, 820bp and 1608bp respectively;

The plaque detected result of Figure 51 encephalitis/Dengue 4 type embedded virus is 5.99lgPFU/ml according to the virus titer that plaque counting obtains;

The growth curve of Figure 52 Japanese encephalitis/dengue chimeric virus in PHK cell;

The Westernblot qualification of Figure 53 Japanese encephalitis/dengue chimeric virus.Result display embedded virus can be observed clear band in dengue virus E protein corresponding position, and encephalitis b virus has no band;

The immunogenicity detected result of Figure 54 encephalitis/Dengue 4 type embedded virus JD4.

Concrete embodiment

Embodiment 1: containing the recovery of the structure of the full-length infectious full length cDNA clone of JEV vaccine strain SA14-14-2, qualification and recombinant virus

One, the transformation of carrier

(1) transformation of low copy plasmid pACNR

Dissolve primer Oligo-1(5'-CGCGCCATTA respectively gGCGCCtTAT aGATCTaATG tCCGGAtTAT gGATCCtGAT tGATCAtTAT tCTAGAaTTAC-3', underscore place is followed successively by KasI, BglII, BspEI, BamHI, BclI, XbaI recognition site, synthetic) and Oligo-2(5'-TCGAGTAAT tCTAGAaTAA tGATCAaTCA gGATCCaTAA tCCGGAcATT aGATCTaTAA gGCGCCtAATGG-3', after forming complementary double-strand with Oligo-1,5' and 3' end forms Ascl and Xhol enzyme respectively and cuts rear cohesive end, synthetic) in 100 μ l sterilizing distilled waters, respectively get 10 μ l to mix, naturally cooling after being heated to 100 DEG C, get 2 μ l with being connected in 4 DEG C with the pACNR of AscI and Xhol double digestion and spend the night, transformed competence colibacillus cell TOP10(TIANGEN Biotech (Beijing) Co., Ltd.), picking ampicillin-resistant clones extracting plasmid does order-checking qualification (Shanghai Ying Weijie base Bioisystech Co., Ltd).

Sequencing result shows: the polyclone district of the implanted plasmid of restriction enzyme site, obtains the pACNR plasmid of polyclone position point through transformation.

(2) transformation of BAC53 carrier

First with carrier pBAC53e3.6 sequence for the following three pairs of primers of stencil design:

BAC53F1：

5-G aAGCTTGGATCCGCATGCGTCGACCTCGAGcAATTCTCATGTTTGACAGCTTATCAT-3(dashed part is the part polyclone restriction enzyme site added, and is followed successively by Hind III, BamH I, Bsiw I, Sal I and Xho I)

BAC53R1：

5-TT gGTACCtCTAGA aCGCGTgGCTTCGCCCTGTCGCTCG-3(dashed part is restriction endonuclease digestion site, Kpn I and Mlu I, and wherein MluI is the restriction enzyme site introduced)

BAC53F2:5-GG aCGCGTtGAGCGAGGAAGCACCAGGGAAC-3(dashed part is people is the restriction endonuclease digestion site Mlu I added)

BAC53R2:5-TGAATATGAAGATCT gGTACCcATCCGTGA-3(dashed part is restriction endonuclease digestion site KpnI)

BAC53F3:5-GG aCGCGTcCTCAATGTATCACGGATG gGTACCaGATCT-3(dashed part is restriction endonuclease digestion site, Mlu I and KpnI, and wherein MluI is the restriction enzyme site introduced)

BAC53R3：

5-GG aAGCTTGGCGCCGGCGCGCCgCTAGC gCGGCCGCaGCGACACACTTGCATCGGATGC-3(dashed part is the part polyclone restriction enzyme site added, and is followed successively by Hind III, Kas I, Asc I and Not I)

Getting 2 μ lpBAC53e3.6 plasmid DNA is template, respectively by primer BAC53F1 and BAC53R1, the corresponding PCR fragment of BAC53F2 and BAC53R2, BAC53F3 and BAC53R3 pairing amplification, and called after BAC53-P1, BAC53-P2, BAC53-P3 respectively.Archaeal dna polymerase used is NEB company of the U.S. super fidelity dna polysaccharase, amplification condition is: 98 DEG C of 2min denaturations; 98 DEG C of 10sec, 58 DEG C of 15sec, 72 DEG C of 1.5min, 30 circulations; 72 DEG C of 10min.The archaeal dna polymerase LA of the DNA fragmentation TaKaRa company of amplification carries out adding A reaction: 10 × LAbuffer1 μ l, dNTP1 μ l, DNA7.5 μ l, LA0.5 μ l, 72 DEG C of 30min. reactions terminate, and pGEM-Teasy carrier that is direct and PROMEGA company carries out ligation.2 × Rapidligationbuffer5 μ l, pGEM-Teasy1 μ l, add the PCR primer 3 μ l of A, T4DNAligase1 μ l, 4 DEG C of connections are spent the night.Connect product conversion competent cell TOP10, be laid on the ampicillin/LB plates containing 100ug/ml, next day, the colourless bacterium colony Zengjing Granule of picking, extracting plasmid carries out order-checking qualification.

By the clone of above-mentioned three fragments correct for order-checking respectively with restriction restriction endonuclease HindIII and MluI, MluI and KpnI, KpnI and HindIII digestions, reclaim fragment BAC53-P1, BAC53-P2, BAC53-P3, T4DNAligase1 μ l, 4 DEG C of connections are spent the night.Connect product conversion competent cell DH10B, the LB be laid on containing 25ug/ml paraxin is dull and stereotyped, next day, and the colourless bacterium colony Zengjing Granule of picking, extracting plasmid carries out order-checking qualification, called after BAC53, and through order-checking, its sequence is as shown in SEQIDNO.1.

Carry out digestion verification with restriction restriction endonuclease Hind III and Kpn I, as shown in Figure 1, restriction enzyme mapping is consistent with theory.

Two, the preparation of JEV attenuated strain SA14-14-2 virus cDNA

(1) extracting of JEV attenuated strain SA14-14-2 viral RNA

JEV vaccine virus (SA14-14-2 strain) is produced by Chengdu Institute of Biological Products Co., Ltd., by specification redissolves, the RNA extraction agent box (HighpureviralRNAkit) that 400 μ l virus liquid Roche companies of getting produce carries out the extracting (by specification carries out) of RNA, RNA be stored in-80 DEG C for subsequent use.

(2) reverse transcription of viral cDNA

The viral RNA (getting 11 μ l respectively) extracted is used two primer RR5 (5'-GACTGCTTCCTGTGATTGCA-3') and RR13 (5'-AGATCCTGTGTTCTTCCTCACCACCAGCTACA-3') retrovirus cDNA respectively, used kit is Invitrogen company SuperScriptTMIIIReverseTranscriptase, primer is synthesized by Shanghai Ying Weijie base Bioisystech Co., Ltd, and method reference reagent box specification sheets carries out.

(3) pcr amplification of viral cDNA fragment and order-checking

Getting viral cDNA2 μ l is template, respectively primers F 1 and R1, F2 and R3, F4 and R4, F5 and R6, F7 and R8, F9 and R10, F11 and the R11 pairing corresponding cDNA fragment of amplification (primer information is in table 1), archaeal dna polymerase used is the PrimeSTAR of TaKaRa company, increase with touchdown PCR (Touch-downPCR), amplification condition is: 98 DEG C of 2min; 98 DEG C of 10sec, 58.5 DEG C → 53.5 DEG C 10sec72 DEG C of kb/min, 10 circulations; 98 DEG C of 10sec53.5 DEG C of 10sec, 72 DEG C of kb/min, 20 circulations; 72 DEG C of 10min.The archaeal dna polymerase LA of the DNA fragmentation TaKaRa company of amplification carries out adding A reaction: 10 × LAbuffer1 μ l, dNTP1 μ l, DNA7.5 μ l, LA0.5 μ l, and 72 DEG C of 30min. reactions terminate, and carrier T pMD19-T that is direct and TaKaRa company carries out ligation.2 × ligationbuffer5 μ l, pMD19-T1 μ l, add the PCR primer 3 μ l of A, ligase1 μ l, 4 DEG C of connections are spent the night.Connecting product, to be laid on LB containing penbritin, IPTG and X-gal dull and stereotyped, next day, the colourless bacterium colony Zengjing Granule of picking, and extracting plasmid carries out enzyme and to cut and check order qualification.

The primer cloned by table 1

Result shows: be template with cDNA, and amplified 7 DNA fragmentations corresponding to theoretical size, size is respectively 0.5kb, 2.2kb, 0.8kb, 2.1kb, 1.5kb, 2.1kb, 1.8kb.Further, all successful clone is to pMD19-T for 7 fragments, and sequencing result shows: except the silent mutation that fragment 1 and 2 people is introducing, all fragments are all without base mutation, insertion and disappearance.

Three, containing JEV5 ' half molecule (base 1-3450) plasmid, containing JEV3 ' half molecule (3445-10977) plasmid and containing the structure of JEV virus full length cDNA carrier

(1) structure containing JEV5 ' half molecule (base 1-3450) plasmid and enzyme cut qualification

By Fig. 1 program 5 ' end 3 amplified fragments (size is respectively 0.5kb, 2.2kb, 0.8kb) be cloned into successively in the pACNR that step one prepares with AscI and KasI, KasI and BglII, BglII and BspEI respectively, obtain containing JEV5 ' half molecule plasmid pJEV5 '.

Result shows: 3 DNA fragmentations varied in size in low copy plasmid, are shown in Fig. 2 by successful clone.

(2) containing structure and the qualification of JEV virus full length cDNA

By method described in Fig. 3,4 DNA fragmentations of increased 3 ' end are spliced and are cloned into corresponding restriction enzyme site in pJEV5 ', obtain the plasmid containing JEV full-length cDNA, called after pJFC, its structural representation is shown in Fig. 3.

With restriction enzyme NotI and XhoI, the encephalitis full-length cDNA on pJFC is cut simultaneously, be inserted in bacterial artificial chromosome (BAC53), be built into JEV full-length clone BAC53-JFC.

With large extraction reagent kit (QIAfilterPlasmidMaxiKit) large quantity extracting plasmid of QIAGEN company, serve the order-checking of Hai Yingweijie base Bioisystech Co., Ltd, and with restriction enzyme, enzyme is carried out to plasmid simultaneously and cut qualification.

Result shows: enzyme cuts result and theory matches (see Fig. 4,5); According to Sequencing chromatogram, the sequence that can read pJFC and this BAC53-JFC is respectively as shown in SEQIDNO.2 and 3, and except the silent mutation of artificial C protein gene the 378th Nucleotide introduced, all the other each several parts of JEV virus are without base mutation, insertion and disappearance.

(3) structure containing JEV3 ' half molecule (3445-10977) plasmid and qualification

With BspEI and Xhol double digestion plasmid pJFC, reclaim the DNA fragmentation of 7.5kb size, be connected with the low copy plasmid pACNR cut by same enzyme, Screening and Identification recon, called after pJEV3 '.

Enzyme is cut and is shown with sequencing result: containing the success of JEV3 ' half molecule (base 3445-10977) plasmid construction.

Four, the in-vitro transcription of JEV full-length infectious CDNA clones, transfection and JEV recovered virus (rJEV) rescue and go down to posterity

After being about 3h in 37 DEG C with Xhol digestion pJFC, add mung-bean nuclease in 30 DEG C of effect 30min, add the SDS deactivation mung-bean nuclease that final concentration is 1g/L, PCR primer reclaims test kit and reclaims linearizing endonuclease bamhi, to reclaim fragment for template, with Promega company of U.S. in-vitro transcription test kit (PromegaRiboMAXLargeScaleRNAProductionSystems-SP6) in-vitro transcription RNA, electrotransfection BHK21 cell, 37 DEG C, the CO of 5% ₂cultivating 5d, to occurring obvious cytopathy, collecting nutrient solution supernatant with freeze-thaw method, called after rJEV.Get the first-generation (P1) viral supernatants and do virus titer mensuration in BHK21 cell.And inoculate BHK21 cell with supernatant, the method for results supernatant carries out viral passages.

Result shows: transfection BHK21 cell 5 days, pathology appears in visible cell.Virus titer is about 6.0lgPFU/ml, and rJEV recovered virus titre that primary hamster kidney cell goes down to posterity can reach 7.5lgPFU/ml, similar to vaccine strain.

Five, the qualification of recombinant virus rJEV

(1) RT-PCR qualification

Get the third generation (P3) viral supernatants, with Roche Holding Ag high purity viral RNA extraction agent box (HighPureViralRNAKit) extracting rJEV viral RNA, use primer

RR5(5'-GACTGCTTCCTGTGATTGCA-3')

With RR13 (5'-AGATCCTGTGTTCTTCCTCACCACCAGCTACA-3 ') retrovirus cDNA, as template, carry out pcr amplification with primers F 1 and R3, F4 and R5, F6 and R7, F8 and R9, F10 and R10, F11 and R11 pairing respectively, serve Hai Yingweijie base Bioisystech Co., Ltd after gained PCR fragment purifying and carry out sequencing.

Result shows: PCR fragment size and theory match, and (size is respectively 2.6kb, 2.1kb, 1.8kb, 1.7kb, 1.0kb, 1.8kb), see Fig. 6, sequencing result shows that C protein gene the 378th bit base contains the artificial silent mutation (A → C) introduced, and all the other do not find base mutation everywhere.

(2) identified by immunofluorescence recovered virus

Inoculation 3 × 10 ⁴individual LLC-MK2 cell is in 96 orifice plates, next day, treat that cell grows up to individual layer, by 103PFU/ hole inoculation P3 virus in hole, in 37 DEG C, the CO2 of 5% cultivates 2 days, suck nutrient solution, PBS washs 1 cell, add methyl alcohol room temperature and fix 20min, discard methyl alcohol, PBS washs 3 times, add 0.2%Tritonx-100 room temperature permeable membrane 10min, PBS washs 1 time, add anti-JEVE protein monoclonal antibody (1:10, Abcam company of the U.S.), hatch 1h for 37 DEG C, PBS washs 3 times, add the anti-(1:100 of sheep anti-Mouse two of FITC mark, SantaCrus company of the U.S.), hatch 1h for 37 DEG C, PBS washs 3 times, fluorescence microscope is also taken pictures, see Fig. 7.

(3) rJEV Plaque Formation on BHK21 cell

BHK21 cell is cultivated in six orifice plates, treat that cytogamy degree reaches about 80%-90%, inoculation P3 recombinant virus and vaccine strain, 37 DEG C of absorption 1h, coverture final concentration is the low melting-point agarose of 10g/L, in 37 DEG C, 5%CO2 cultivates after 5d and detects virus titer, plaque form and size by the violet staining of 10g/L.

Result shows: rJEV with JEV vaccine strain SA14-14-2 can form the similar plaque of size and form on BHK21 cell, sees Fig. 8.

(4) detection of rJEV neurovirulence

Respectively by recovered virus inoculation BALB/c mouse in 4 week age, 0.03ml(is containing viral 3.5lgPFU)/only, and 3-5 days BALB/c suckling mouses, 0.02ml(is containing viral 3.3lgPFU)/only, each 10, raise 14 days, observe Viral nerve virulence.

Experimental result is in table 2

Table 2 recovered virus neurovirulence detects

Experimental result shows, the present invention successfully constructs the infection and encephalitis B virus infection sex clone containing JEV full-length cDNA: pJFC and BAC53-JFC, use this infections clone to obtain recovered virus, recovered virus can grow on BHK21 cell, can as gene framework.

Embodiment 2: the Preparation and identification of encephalitis/Dengue 1 type embedded virus

One, the preparation of encephalitis/Dengue 1 type embedded virus

1, the synthesis of dengue virus 1 type antigen gene prM-E fragment

Get the gene fragment prME(of dengue virus 1 type ThD1000881 strain coding E protein as shown in SEQIDNO.4), after the Nucleotide in the corresponding site of 9 Nucleotide encephalitis b virus SA14-14-2 strain of E protein carboxyl terminal albumen of encoding in this fragment is replaced, merge with N-terminal 204 Nucleotide of encephalitis b virus SA14-14-2 strain NS1 protein gene.

Then this fragment is inserted into pcDNA3.1 carrier, obtains the plasmid clone pcDNA3.1-D1PrME of dengue virus 1 type antigen gene prM-E.

2, the structure of encephalitis/Dengue 1 type 5 ' half molecule

By the above-mentioned pcDNA3.1-D1prM-E plasmid containing dengue virus 1 type antigen gene prM-E with BglII single endonuclease digestion, remove the part (BglII13-BglII900) between pcDNA3.1-D1prM-E plasmid two BglII restriction enzyme sites, the QiaquickGelextractionkit of QIAGEN company is adopted to reclaim 6.7kb fragment, make this fragment from connecting and transforming TOP10 competent cell with the T4DNAligase of NEB company of the U.S. again, selected clone carries out the extraction of plasmid a small amount of with the Qiaprepspinminiprepkit of QIAGEN company and connects and carry out digestion verification, the plasmid called after pcDNA3.1-D1prM-E (MOD) obtained.

With restriction restriction endonuclease NotI and KasI double digestion pJEV-5 ' plasmid and pcDNA3.1-D1prM-E (MOD) plasmid respectively, glue reclaims the fragment of the 728bp after pJEV-5 ' plasmid enzyme restriction, by its with NotI with KasI double digestion after pcDNA3.1-D1prM-E (MOD) plasmid be connected, called after pJD1-2650.

And then with BglII single endonuclease digestion pJEV-5 ' plasmid and pJD1-2650 plasmid, wherein reclaim the short-movie section of the 1241bp of BglII single endonuclease digestion pJFC plasmid, be inserted in the pJD1-2650 plasmid of BglII single endonuclease digestion, the plasmid that digestion verification 1241 fragment forward inserts, thus obtain 5 ' half chimeric molecular cloning of encephalitis/Dengue 1 type, called after pJD1-5 ' (Fig. 9 and 10).

3, the structure of encephalitis/Dengue 1 type full-length clone

With restriction enzyme NotI and the above-mentioned pJD1-5 ' of BspEI double digestion, reclaim 3.7kb fragment, the plasmid clone pJFC of encephalitis full-length cDNA is contained with BspEI and XhoI double digestion, reclaim 7.5kb fragment, carry out Ligation in vitro with T4DNAligase to two fragments, glue reclaims and connects product (11.2kb).

That reclaims with NotI with XhoI double digestion is connected product and again reclaims, use NotI and XhoI double digestion BAC53 plasmid simultaneously, reclaim 6.4kb fragment, two fragments are connected with T4DNAligase, transform TOP10 competent cell, a small amount of extraction plasmid carries out enzyme and cuts qualification and order-checking, obtains the plasmid containing encephalitis/Dengue 1 type chimaeric full length and clone, called after pJD1FC.

Enzyme cuts result as shown in Figure 11,12 and 13, according to Sequencing chromatogram, can read pJD1FC sequence as shown in SEQIDNO.8.

4, the preparation of the external transcript RNA of encephalitis/Dengue 1 type embedded virus

By the XhoI restriction enzyme linearizing of pJD1FC plasmid, with the PureLinkPCRpurificationkit purified linear product of Invitrogen, the RibomaxlargescaleRNAproductionsystem-SP6 of Promega company is then adopted to carry out in-vitro transcription.40 μ l reaction systems comprise: 5 × transcriptbuffer8 μ l, rATP(100mM) 2 μ l, rCTP(100mM) 2 μ l, rUTP(100mM) 2 μ l, rGTP(100mM) 0.7 μ l, m7Gcapanalog(40mM) 3 μ l, enzymemix4 μ l, linearizing product 18.3 μ l.After reaction system hatches 4h in 37 DEG C, add 2 μ lRNase-freeDNase in 37 DEG C of digestion template 30min, then use the RneaseMinikit purifying RNA product of QIAGEN company, save backup (Figure 14) in-70 DEG C.

5, the rescue of encephalitis/Dengue 1 type embedded virus

With pancreatin dispersion BHK21 cell, the centrifugal 5min of 800rpm, with precooling PBS washed cell 2 times, with 160 μ lPBS re-suspended cells, add the PBS that the above-mentioned RNA(compared with control cells of 5-10 μ g adds same volume), mixing, with voltage 140V, burst length 25ms, pulse 1 time, carries out electricity with the GenepulserXcell of Bio-Rad and turns.Then cell is placed in 6min on ice, then proceeds in MEM growth media, 37 DEG C of cultivations.Nutrient solution is replaced by MEM maintenance medium by next day, and continue cultivation 4 ~ 6 days, obvious CPE appears in experimental group BHK21 cell, and compared with control cells is without pathology (Figure 15).By sick cell and supernatant liquor multigelation 3 times, the centrifugal 10min of 4000rpm, results supernatant liquor, in-70 DEG C of preservations after packing, the first-generation embedded virus called after JD1(P1 of acquisition).

6, the Secondary Culture of encephalitis/Dengue 1 type embedded virus

Get above-mentioned embedded virus JD10.5ml and inoculate primary hamster kidney cell (PHK cell), after 37 DEG C of absorption 1h, add MEM substratum, be cultured to cell and occur CPE.Aspirate supernatant, the centrifugal 10min of 4000rpm, the supernatant liquor of results is s-generation embedded virus JD1(P2), in-70 DEG C of preservations after packing.By the same way JD1 was uploaded to for the 10th generation at PHK cell, every qualification (Figure 16) is carried out to embedded virus therebetween.

Two, the discriminating of encephalitis/Dengue 1 type embedded virus

1, the RT-PCR qualification of encephalitis/Dengue 1 type embedded virus

S-generation embedded virus JD1(P2 is extracted with the HighpureviralRNAkit of Roche) RNA, and with the Super of Invitrogen embedded virus RNA reverse transcription is cDNA by IIIReverseTranscriptase.With this cDNA for template, carry out pcr amplification with the PrimeSTARHSDNApolymerase of TaKaRa.Primer sequence is as follows: first is right: upstream primer 5'-ACCAACATTGGACATTGAACTCT-3', downstream primer 5'-GGTCCGGACCAGTCTAGTGACAGATCTGACTC-3', product length 1570bp; Second pair: upstream primer 5'-GTGAGGACACAATGACTTAC-3', downstream primer 5'-GGTCCGGACCAGTCTAGTGACAGATCTGACTC-3', product length 2048bp; 3rd is right: upstream primer 5'-TTGGCGCGCCATTTAGGTGACACTATAGAGAAGTTTATCTGTGTGAACTTCTT-3', downstream primer 5'-GTTGGCTGTTATCACTCTCC-3', product length 2067bp.Amplified production all meets expection (Figure 17).

2, the plaque of encephalitis/Dengue 1 type embedded virus detects

BHK21 cell is seeded to six orifice plates, carries out plaque detection.Using MEM nutrient solution as diluent, respectively 10 times of dilutions are carried out to each generation embedded virus.Add each dilution virus liquid 400 μ l respectively, in 37 DEG C of absorption 60min.1% low melting-point agarose 2ml is added, in 37 DEG C of cultivations after to be solidified in every hole.5 days afterwards every hole add 4% paraformaldehyde 2ml and fix 60min, with the dyeing of Viola crystallina dye liquor, observe plaque form and also calculate virus titer (Figure 18) according to plaque quantity.

3, the Westernblot qualification of encephalitis/Dengue 1 type embedded virus

Third generation embedded virus JD1(P3) add 20 μ l5 × proteinloadingbuffer in 80 μ l, boiling water boiling 10min after mixing, the centrifugal 1min of 10000rpm, get supernatant liquor and carry out SDS-PAGE electrophoresis, protein delivery is to nitrocellulose filter, close 1h with 5% skimmed milk room temperature, then use dengue virus 1 type E protein polyclonal blood antibody in 4 DEG C of overnight incubation.PBS washes film, then with the goat anti-rabbit igg incubated at room 1.5h of AP mark.Develop the color with BCIP/NBT alkaline phosphatase colouring reagents box (the green skies, Shanghai) after PBS washes film.Result display embedded virus can be observed clear band at 55kD place, and in the same size with dengue virus E protein, result meets expection (Figure 19).

4, the growth characteristics analysis of encephalitis/Dengue 1 type embedded virus

By MEM maintenance medium by JD1(P2) virus liquid dilution, be 0.1,0.01,0.001 infection with MOI, PHK cell, in 37 DEG C of absorption 120min.Add MEM maintenance medium 20ml, in 37 DEG C of cultivations.Extracted 0.5ml supernatant liquor every 12 hours ,-70 DEG C for subsequent use, supplies volume with 0.5mlMEM maintenance medium, and sample collecting is to inoculating latter 96 hours.Plaque method measures the virus titer of each time point sample of different MOI, draws the growth curve (Figure 20) of JD1 virus in PHK cell.

Result display 0.001-0.01MOI is between the best infective dose of virus, can gather in the crops the virus liquid of the highest virus titer.

5, the immunogenicity experiments of encephalitis/Dengue 1 type embedded virus

(1) immunogen preparation

Third generation encephalitis/Dengue 1 type embedded virus of PHK cell cultures is used as immunogen.With MEM nutrient solution (Gibco), virus quantity is adjusted to 5logPFU/ml for subsequent use.

(2) laboratory animal and grouping

60 female 4 week age BALB/c mouse provided by Chengdu Institute of Biological Products Co., Ltd..Mouse is divided into 2 groups at random, often organizes 30: (1) JD1 embedded virus immune group; (2) negative control group (MEM basic medium).

(3) immunization protocol

Get above-mentioned virus liquid and MEN nutrient solution, fully after mixing, respectively by peritoneal immunity mouse, 500 μ l/ only.After initial immunity, 2 weeks with identical approach and dosage booster shot once.Initial immunity starts blood sampling after 3 weeks, after this every 2 weeks 1 time to the 13rd week is terminated, each 5, amounts to 6 times.Separation of serum, 56 DEG C of deactivations 30 minutes after diluting 10 times with PBS ,-20 DEG C save backup.

(4) inspection of immune serum NAT

Above-mentioned serum sample (1:10) serum dilution (PBS) is carried out doubling dilution to 1:640 according to 1:2, get every extent of dilution sample 200ul respectively to contain 50-100 dengue virus 1 type with same volume and mix, with 1 hour in 37 DEG C, join and cover with on six orifice plates of BHK21 cell, cultivate after 5 days, with violet staining for 37 DEG C.Negative control is done not add sero-fast MEM substratum.To reduce the serum-concentration of the most high dilution of 50% virus plaques number as NAT.

Result shows, after encephalitis/Dengue 1 type embedded virus immune mouse, can produce the neutralizing antibody of special anti-homotype dengue virus, antibody titer can reach 1:34.82(and see Figure 21).

Experimental result illustrates, the present invention successfully builds and obtains encephalitis/Dengue 1 type embedded virus, and this embedded virus can massive duplication, and can grow on BHK21 cell, the NAT of generation is high, can be prepared as dengue vaccine.

Structure, the virus of embodiment 3 encephalitis/Dengue 2 type embedded virus full-length cDNA plasmid are recovered and important biomolecule CHARACTERISTICS IDENTIFICATION

One, the structure of encephalitis/Dengue 2 type embedded virus full-length cDNA plasmid

1, the synthesis of DEN2prM/E protein coding gene

Get the gene fragment prM-E(of DEN2PUO-218 strain coding E protein as shown in SEQIDNO.5), after the Nucleotide in the corresponding site of 9 Nucleotide encephalitis b virus SA14-14-2 strain of E protein carboxyl terminal albumen of encoding in this fragment is replaced, 204 Nucleotide held with encephalitis b virus SA14-14-2NS1 protein gene 5 ' merge, be inserted in carrier pMD18-T, for the structure of chimaeric full length clone.

2, the structure of encephalitis/Dengue 2 type Chimeric fragment 5 ' half molecule plasmid pJE/DEN2-5 '

The gene fragment prM/E(of synthesis is comprised N-terminal 204 Nucleotide of coding JEVNS1) cut with restriction restriction endonuclease KasI and BglII enzyme, reclaim the DNA fragmentation of 2.1kb, simultaneously with identical two restriction restriction endonuclease cutting 5 ' half molecular cloning pJEV5 ', and reclaim large fragment (4.0kb), two fragments are connected and transformed competence colibacillus cell TOP10, select positive colony and carry out enzyme and to cut and check order qualification, and called after pJD2-5 '.

3, the structure of encephalitis/Dengue 2 type full-length cDNA plasmid and qualification

Encephalitis/Dengue 2 type 5 ' half molecule plasmid pJE/DEN2-5 ' of structure and encephalitis 3 ' half molecule plasmid pJEV3 ' is cut with restriction restriction endonuclease BspEI and XhoI enzyme simultaneously, reclaiming size is respectively the DNA fragmentation of 6.1kb and 7.5kb, connect and transformed competence colibacillus cell TOP10, select positive colony and carry out plasmid extraction, digestion verification and total length order-checking qualification, called after pJD2FC.

Enzyme cuts result as shown in figure 22, according to Sequencing chromatogram, can read the sequence of pJD2FC as shown in SEQIDNO.9.

4, the in-vitro transcription of viral RNA, transfection and embedded virus acquisition and go down to posterity

After digesting pJD2FC3h in 37 DEG C with Xhol, add mung-bean nuclease in 30 DEG C of effect 30min, add the SDS deactivation mung-bean nuclease that final concentration is 1g/L, PCR primer reclaims test kit and reclaims linearizing endonuclease bamhi, to reclaim fragment for template, with the outer transcribe rna of in-vitro transcription kit box, and reclaim test kit recovery RNA with RNA, after electrotransfection BHK21 cell (electrotransfection condition is 140 volts of voltages), be transferred in T-25 culturing bottle, in 37 DEG C, the CO2 of 5% cultivates 5d, collects nutrient solution supernatant, called after JD2 by the method for multigelation.Detect the virus in supernatant by plaque detection method, and carry out viral passages with 0.5ml supernatant inoculation PHK cell.

Result shows: transfection BHK21 cell 5 days, has no cell and occur pathology.But can virus be detected in supernatant, virus titer is about 3log10PFU/ml.With supernatant inoculation PHK cell, PHK cells showed cytopathic effect (Figure 23) can be made at about 30h.

Two, the qualification of encephalitis/Dengue 2 type embedded virus

1, the RT-PCR qualification of encephalitis/Dengue 2 type embedded virus

Get p3 for viral supernatants, with high purity viral RNA extraction agent box extracting embedded virus RNA, with primer RR5 (5'-GACTGCTTCCTGTGATTGCA-3 ') and RR13 (5'-AGATCCTGTGTTCTTCCTCACCACCAGCTACA-3 ') retrovirus cDNA, as template, carry out pcr amplification with primers F 1 and R3, F4 and R5, F6 and R7, F8 and R9, F10 and R10, F11 and R11 pairing respectively, serve Hai Yingweijie base Bioisystech Co., Ltd after gained PCR fragment purifying and carry out sequencing.

Result shows: PCR fragment size and theory match (is respectively 2.6kb, 2.1kb, 1.8kb, 1.7kb, 1.0kb, 1.8kb) (see Figure 24), sequencing result shows that C protein gene the 378th bit base has the artificial silent mutation (A → C) introduced, and all the other do not find base mutation everywhere.

2, identified by immunofluorescence embedded virus JD2

Inoculate 3 × 104 vero cells in 96 orifice plates, next day, treat that cell grows up to individual layer, by 103PFU/ hole inoculation p3 generation virus in hole, in 37 DEG C, the CO2 of 5% cultivates 2 days, suck nutrient solution, PBS washs 1 cell, add methyl alcohol room temperature and fix 20min, discard methyl alcohol, PBS washs 3 times, add 0.2%Tritonx-100 room temperature permeable membrane 10min, PBS washs 1 time, add anti-JEVE protein monoclonal antibody (Abcam Products, 1:10 dilutes), hatch 1h for 37 DEG C, PBS washs 3 times, add anti-(the SantaCruz Products of sheep anti-Mouse two of FITC mark, 1:100 dilutes), hatch 1h for 37 DEG C, PBS washs 3 times, inverted fluorescence microscope (Olympus Products) is observed and takes pictures, the results are shown in Figure 25.

3, JD2 Plaque Formation and at PHK cell growth status on BHK21 cell

Digestion BHK21 cell, treat that cytogamy degree reaches about 80%-90%, p3 is for recombinant virus and vaccine strain in inoculation, 37 DEG C of absorption 1h, coverture final concentration is the low melting-point agarose of 10g/L, in 37 DEG C, 5%CO2 cultivates violet staining detection virus titer, plaque form and size (see Figure 26) with 10g/L after 5d.Inoculate PHK cell with different m.o.i., every 12 hr collections viral supernatants, detect virus titer, draw growth curve (Figure 27).

Result shows: DEN2 can form less plaque on BHK21 cell, and rJEV with JEVSA14-14-2 forms the similar plaque of size, and the plaque that JD2 is formed is similar to rJEV.

Show at the growth curve of PHK cell: inoculate the virus liquid that lower m.o.i. is more conducive to collect higher titre, and higher m.o.i. inoculates PHK, the peak of virus appears at about 24h, and virus titer is also lower, declines also very fast.

4, the immunogenic detection of JD2

With 0.5ml virus liquid (containing 4.4log10PFU) intraperitoneal inoculation kunming mice in 4 week age 30, two Zhou Houyong similarly measure booster immunization and once (contrast with MEM) simultaneously, from the 4th week, 5 mouse are put to death every 2 weeks, collect serum, reduce Neutralizing test (plaquereductionneutralizationtest, PRNT) with plaque and detect anti-DEN2 NAT.First two-fold dilution's antiserum(antisera) (from 1:10), then often pipe adds 100PFU virus in 37 DEG C and 1h, virus inoculation and antiserum(antisera) mixed solution are to individual layer BHK21 cell adsorption 1h, supernatant discarded, add maintenance medium containing 10g/L low melting-point agarose and 2% new-born calf serum in 37 DEG C, the CO2 of 5% cultivates 5d, plaque dyes, count the plaque number in each hole, in calculating and titre, get the most highly diluted multiple of 50% neutralising capacity as sero-fast Neutralizing titer, its NAT can reach 1:92(and see Figure 28).

5, JD2 immunity is studied mouse immune provide protection

With 0.5mlp3 virus liquid (containing 4.4log ₁₀pFU) peritoneal immunity kunming mice in 4 week age, respectively at immunity the 2nd week and the 4th week with DEN2(NGC) the brain endoadaptation strain encephalocoele side of inject attacks, and contrast with MEM, the rear observation of attack 14 days, adds up the mortality ratio (the results are shown in Table 4) of mouse simultaneously.

Table 4JD2 immune effect

After result shows the immunity of JD2 embedded virus, mouse invasion rate is far smaller than the sickness rate of control group.

Experimental result explanation; the present invention successfully builds and obtains encephalitis/Dengue 2 type embedded virus; its can in PHK cell massive duplication; the neutralizing antibody of dengue virus can be produced; produce the titre of the neutralizing antibody of dengue virus up to 1:92; active immunity provide protection is strong, can be prepared as dengue vaccine.

The Preparation and identification of embodiment 4 encephalitis/Dengue 3 type embedded virus

One, the preparation of encephalitis/Dengue 3 type embedded virus

1, the synthesis of dengue virus 3 type antigen gene prM-E fragment

Get the gene fragment prM-E(of dengue virus 3 type PaH881-88 strain coding E protein as shown in SEQIDNO.6), after the Nucleotide in the corresponding site of 9 Nucleotide encephalitis b virus SA14-14-2 strain of E protein carboxyl terminal albumen of encoding in this fragment is replaced, merge with N-terminal 204 Nucleotide of encephalitis b virus SA14-14-2 strain NS1 protein gene, this fragment is inserted into pMD18-T carrier, obtains the plasmid clone pMD-D3prM-E of dengue virus 3 type antigen gene prM-E.

2, the structure of encephalitis/Dengue 3 type 5 ' half molecule

By pMD-D3prM-E after NEB company Kas I and Bgl II double digestion, the QiaquickGelextractionkit of Qiagen is adopted to reclaim small segment (2.1kb), double digestion is carried out to encephalitis 5 ' half molecular cloning pJEV-5 ' restriction enzyme KasI and BglII simultaneously, and reclaim large fragment (4.0kb), two fragments are connected with the T4DNAligase of NEB and transforms TOP10 competent cell, the Qiaprepspinminiprepkit of selected clone Qiagen carries out plasmid and extracts in a small amount, and digestion verification and order-checking are carried out to plasmid, called after pJD3-5 ', find after order-checking to have lacked 8 bases (Figure 29) at E gene 1440-1448 place.Be that template adopts fusion DNA vaccine method 8 bases to disappearance to repair with pJD3-5 ' plasmid, with Invitrogen PureLinkPCRpurificationkit purified pcr product (about 3.5kb) (Figure 30), fragment is reclaimed with after NEBAsc I and BspE I double digestion, the small segment (about 2.1kb) reclaimed through BspE I and BamH I double digestion with pJEV-3 ' plasmid, connect with the T4DNAligase of NEB, large fragment (5.7kb) is reclaimed with the QiaquickGelextractionkit of Qiagen, again this is reclaimed fragment to be connected with the BAC53 carrier recovery fragment (about 6.4kb) through NEBAsc I and BamH I double digestion, transform DH10B competent cell, the Qiaprepspinminiprepkit of selected clone Qiagen carries out plasmid and extracts in a small amount, and digestion verification and order-checking are carried out to plasmid, obtain encephalitis/Dengue 3 type 5 ' half molecular cloning, called after BAC53JD3-5 ' (Figure 31).

3, pJEV-3 ' half(Sal I) structure of half molecule

PCR method is adopted to increase from Vaccinum Encephalitidis Epidemicae strain (SA14-14-2) Xba I to 3 ' end sequence (fragment F11), at 3 ' end introducing Sal I and Xho I restriction enzyme site.Fragment is reclaimed by after PCR primer NEBXba I and Xho I double digestion, with by the carrier segments part reclaimed after Xba I and Xho I double digestion pMDJEV-3 ' half, after connecting with the T4DNAligase of NEB, transform TOP10 competent cell, the Qiaprepspinminiprepkit of selected clone Qiagen carries out plasmid and extracts in a small amount, and digestion verification and order-checking are carried out to plasmid, obtain encephalitis/Dengue 3 type 5 ' half molecular cloning, called after pJEV-3 ' half(Sal I) (Figure 32,33).

4, the structure of encephalitis/Dengue 3 type chimaeric full length clone

Encephalitis/Dengue 3 type 5 ' half molecular cloning BAC53JD3-5 ' of above-mentioned structure and JEV3 ' half molecule pJEV-3 ' (Sal I) restriction restriction endonuclease BamH I and SalI is carried out double digestion, reclaim large fragment (being about 12kb and 5.4kb respectively) respectively, carry out connecting and transform DH10B competent cell, selected clone carries out plasmid extraction in a small amount, digestion verification and total length order-checking, obtain encephalitis/Dengue 3 type full length cDNA clone, called after pJD3FC.

PJD3FC enzyme cuts result as shown in figs. 34 and 35, completely the same with expection; According to Sequencing chromatogram, the nucleotide sequence of pJD3FC can be read as shown in SEQIDNO.10.

5, the preparation of the external transcript RNA of encephalitis/Dengue 3 type embedded virus

By BAC53JD3FC restriction enzyme SalI linearizing, with the PureLinkPCRpurificationkit purified linear product of Invitrogen, the RibomaxlargescaleRNAproductionsystem-SP6 of Promega is then adopted to carry out in-vitro transcription.40 μ l reaction systems comprise: 5 × transcriptbuffer8 μ l, rATP(100mM) 2 μ l, rCTP(100mM) 2 μ l, rUTP(100mM) 2 μ l, rGTP(100mM) 0.7 μ l, m7Gcapanalog(40mM) 3 μ l, enzymemix4 μ l, linearizing product 18.3 μ l.After reaction system hatches 4h in 37 DEG C, add 2 μ lRNase-freeDNase in 37 DEG C of digestion template 30min, then use the RneaseMinikit purifying RNA product of Qiagen, save backup (Figure 36) in-70 DEG C.

6, the rescue of encephalitis/Dengue 3 type embedded virus

By the BHK21 cell trysinization of adherent growth, cell is blown and beaten with the MEM substratum (Gibco) containing 10% calf serum, the centrifugal 5min of 800rpm, with aseptic precooling PBS washed cell 2 times, with 160 μ lPBS re-suspended cells, the in-vitro transcription product RNA(compared with control cells adding the above-mentioned purifying of 5-10 μ g adds the PBS of same volume), carry out electricity according to the GenepulserXcell of the experiment parameter Biorad of voltage 140V, burst length 25ms, pulse 1 time after mixing and turn.Then cell is placed in 6min on ice, then proceeds to 37 DEG C of cultivations in the MEM substratum containing 10% calf serum.Substratum is replaced by the MEM substratum containing 2% calf serum by next day, continues to be cultured to 5th ~ 7 days to occurring cytopathy.By transfectional cell and supernatant liquor multigelation 3 times, the centrifugal 10min of 4000rpm, results supernatant liquor, in-70 DEG C of preservations after packing, the first time embedded virus called after JD3(P1 of acquisition).

7, the Secondary Culture of encephalitis/Dengue 3 type embedded virus

The first-generation embedded virus JD30.5ml getting above-mentioned acquisition inoculates the primary hamster kidney cell (PHK cell) of adherent growth, after 37 DEG C of absorption 1h, adds the MEM substratum containing 2% calf serum, continues to be cultured to cell and occur CPE.Aspirate supernatant, the centrifugal 10min of 4000rpm, the supernatant liquor of results is s-generation embedded virus JD3(P2), in-70 DEG C of preservations after packing.By the same way JD3 was uploaded to for the 15th generation at PHK cell, every qualification (Figure 37) is carried out to embedded virus therebetween.

Two, the qualification of encephalitis/Dengue 3 type embedded virus

1, the RT-PCR of encephalitis/Dengue 3 type embedded virus identifies and order-checking

Third generation embedded virus JD3(P3 is extracted with the HighpureviralRNAkit of Roche company) RNA, and with the Super of Invitrogen embedded virus RNA reverse transcription is cDNA by IIIReverseTranscriptase.With this cDNA for template, carry out pcr amplification with the PrimeSTARHSDNApolymerase of TaKaRa.The primer is that the prM/E of dengue virus 4 kinds of serotypes detects primer, the following D1 of primer sequence: upstream primer 5'-ACCAACATTGGACATTGAACTCT-3 ', downstream primer 5'-AGGTGGTTCTGCCTCAATGT-3 ', product length 1026bp; D2: upstream primer 5'-TCGCTCCTTCAATGACAAT-3', downstream primer 5'-CCTGTGAGTGCTGTGTGC-3', product length 814bp; D3: upstream primer 5'-CCACGGACAGGTTTGGATT-3 ', downstream primer 5'-GAACATCTTCCCAATCGAGCT-3', product length 615bp; D4: upstream primer 5'-CACAGCATGGGACAACAGTG-3', downstream primer 5'-CGTGCCAATCCACAACACT-3', product length 461bp.

Result display only has D3 primer can amplify object band (Figure 38).PCR primer is checked order, consistent with embedded virus expected sequence.

2, the plaque of encephalitis/Dengue 3 type embedded virus detects

BHK21 cell is seeded to six orifice plates, when cytogamy degree reaches 70-80%, carries out plaque detection.Using serum-free MEM substratum as diluent, respectively 10 times of gradient dilutions are carried out to each generation embedded virus.Suck the substratum of BHK21 cell, in each hole, add the virus liquid after 400 μ l dilutions respectively, in the soft jolting of 37 DEG C of absorption 60min, every 20min once.Suck virus liquid after having adsorbed, in every hole, add 2ml1% low melting-point agarose (containing 2% calf serum), in 37 DEG C of cultivations after to be solidified.5 days afterwards every hole add 2ml4% paraformaldehyde and fix 60min, then with the dyeing of Viola crystallina dye liquor, observe plaque form and also calculate virus titer (Figure 39) according to plaque quantity.

3, the drafting of encephalitis/Dengue 3 type embedded virus growth curve

With containing the MEM maintenance medium (Gibco) of 2% calf serum by JD3(P3) to be diluted to infection multiplicity (MOI) be respectively 0.1,0.01,0.001 to virus liquid, the PHK cell of adherent growth in inoculation T75 bottle, in the soft jolting of 37 DEG C of absorption 120min, every 20min once.Suck virus liquid after having adsorbed, add the MEM maintenance medium (Gibco) of 20ml containing 2% calf serum, in 37 DEG C of cultivations.Extracted 0.5ml supernatant liquor every 12 hours ,-70 DEG C of preservations are to be measured, supply volume with 0.5mlMEM maintenance medium, and sample collecting is to inoculating latter 96 hours.Carry out titer determination according to each time point sample of plaque method to different MOI, draw the growth curve (Figure 40) of JD3 virus in PHK cell.The virus titer that result shows different MOI inoculum size rises rapidly after inoculation, and 48-60h reaches the highest after inoculation, after this declines rapidly.The virus titer of high MOI inoculum size occurs that peak value comparatively early, and MOI is that the virus titer of 0.001 inoculum size occurs that peak value is more late, and titre is the highest.

4, the Westernblot qualification of encephalitis/Dengue 3 type embedded virus

At 80 μ l third generation embedded virus JD3(P3) in add 20 μ l5 × proteinloadingbuffer, boiling water boiling 10min after mixing, the centrifugal 1min of 10000rpm, get supernatant liquor and carry out SDS-PAGE electrophoresis, protein delivery is to nitrocellulose filter, close 1h with 5% skimmed milk room temperature, then use dengue virus 3 type E protein polyclonal blood antibody (extent of dilution 1:40 is prepared voluntarily) in 4 DEG C of overnight incubation.PBS washes film, then with goat anti-rabbit igg (extent of dilution 1:5000, the green skies, Shanghai) the incubated at room 1.5h of AP mark.Develop the color with BCIP/NBT alkaline phosphatase colouring reagents box (the green skies, Shanghai) after PBS washes film.According to the same terms, encephalitis b virus JEV (P3) is detected, in contrast.

Result display encephalitis b virus has no band, and embedded virus can be observed clear band at 55kD place, corresponding with dengue virus E protein position, and result meets expection (Figure 41).

5, the immunogenicity experiments of encephalitis/Dengue 3 type embedded virus

(1) immunogen preparation

Gather in the crops the third generation encephalitis/Dengue 3 type embedded virus JD3 supernatant cultivated in PHK cell, measure after virus titer through plaque method, with MEM basic medium (Gibco), virus titer is adjusted to 105/ml for subsequent use.

(2) laboratory animal and grouping

60 Healthy female BALB/c mouse in 4 week age is provided by Chengdu Institute of Biological Products Co., Ltd..Laboratory animal is divided into 2 groups at random, often organizes 30: (1) JD3 embedded virus immune group; (2) negative control group (MEM basic medium).

(3) immunization protocol

Get above-mentioned virus liquid and negative controls, fully pass through abdominal injection approach Mice Inoculated after mixing, 500ul/ only.After initial immunity, 2 weeks with identical approach and dosage booster shot once.Initial immunity starts blood sampling after 3 weeks, after this every 2 weeks 1 time to the 13rd week is terminated, each 5, amounts to 6 times.Separation of serum, 56 DEG C of deactivations 30 minutes after diluting 10 times with PBS ,-20 DEG C save backup.

(4) inspection of immune serum NAT

Above-mentioned serum sample (1:10) serum dilution (PBS) is carried out doubling dilution to 1:640 according to 1:2, every concentration samples is got 200ul respectively and is contained 50-100PFU dengue virus 4 type with same volume and mix, after hatching 1 hour in 37 DEG C after mixing, join and cover with on six orifice plates of BHK21 cell, measure NAT.Negative control is done not add sero-fast MEM substratum.Using can in and the serum-concentration of most high dilution of 1/2 virus as NAT.

Result shows, after encephalitis/Dengue 3 type embedded virus immune mouse, can produce the neutralizing antibody of special anti-homotype dengue virus, NAT is up to 1:56.56 (see Figure 42.

Description of test, the present invention successfully builds and obtains encephalitis/Dengue 3 type embedded virus, its can in PHK cell massive duplication, can produce the neutralizing antibody of dengue virus, immunogenicity is strong, can be prepared as dengue vaccine.

The Preparation and identification of embodiment 5 encephalitis/Dengue 4 type embedded virus

One, the preparation of encephalitis/Dengue 4 type embedded virus

1, the synthesis of dengue virus 4 type antigen gene prM-E fragment

The gene fragment prM-E(SEQIDNO.7 getting dengue virus 4 type 814669 strain coding E protein is shown) by after the replacement of the Nucleotide in the corresponding site of 9 Nucleotide encephalitis b virus SA14-14-2 strain of E protein carboxyl terminal albumen of encoding in this fragment, merge with N-terminal 204 Nucleotide of encephalitis b virus SA14-14-2 strain NS1 protein gene, then this fragment is inserted into TOPO carrier, obtains the plasmid clone TOPO-D4prM-E of dengue virus 4 type antigen gene prM-E.

2, the structure of encephalitis/Dengue 4 type 5 ' half molecule

The above-mentioned TOPO-D4prM-E plasmid containing dengue virus 4 type prM-E sequence is limited restriction endonuclease KasI and BglII double digestion, the QiaquickGelextractionkit of QIAGEN company is adopted to reclaim small segment (2.1kb), double digestion is carried out to encephalitis 5 ' half molecular cloning pJEV-5 ' restriction enzyme KasI and BglII simultaneously, and reclaim large fragment (4.0kb), two fragments are connected with the T4DNAligase of NEB and transforms TOP10 competent cell, the Qiaprepspinminiprepkit of selected clone QIAGEN company carries out plasmid and extracts in a small amount, and digestion verification and order-checking are carried out to plasmid, obtain encephalitis/Dengue 4 type 5 ' half molecular cloning, called after pJD4-5 ' (Figure 43 and 44).

3, the structure of encephalitis/Dengue 4 type full-length clone

Encephalitis/Dengue 4 type 5 ' half molecular cloning pJD4-5 ' of above-mentioned structure and encephalitis 3 ' half molecular cloning pJEV-3 ' half is limited restriction endonuclease BspEI and XhoI and carries out double digestion, reclaim large fragment (being respectively 6.1kb and 7.5kb) respectively, carry out connecting and transform TOP10 competent cell, selected clone carries out plasmid extraction in a small amount, digestion verification and total length order-checking, obtain encephalitis/Dengue 4 type full length cDNA clone, called after pJD4FC.

Enzyme cuts result as Figure 45 and 46, according to Sequencing chromatogram, can read the sequence of pJD4FC as shown in SEQIDNO.11.

4, the preparation of the external transcript RNA of encephalitis/Dengue 4 type embedded virus

By pJD4FC restriction enzyme XhoI linearizing, with the PureLinkPCRpurificationkit purified linear product of Invitrogen, the RibomaxlargescaleRNAproductionsystem-SP6 of Promega company is then adopted to carry out in-vitro transcription.40 μ l reaction systems comprise: 5 × transcriptbuffer8 μ l, rATP(100mM) 2 μ l, rCTP(100mM) 2 μ l, rUTP(100mM) 2 μ l, rGTP(100mM) 0.7 μ l, m ⁷gcapanalog(40mM) 3 μ l, enzymemix4 μ l, linearizing product 18.3 μ l.After reaction system hatches 4h in 37 DEG C, add 2 μ lRNase-freeDNase in 37 DEG C of digestion template 30min, then use the RneaseMinikit purifying RNA product of QIAGEN company, save backup (Figure 47) in-70 DEG C.

5, the rescue of encephalitis/Dengue 4 type embedded virus

By the BHK21 cell trysinization of adherent growth, cell is blown and beaten with the MEM substratum (Gibco) containing 10% calf serum, the centrifugal 5min of 800rpm, with aseptic precooling PBS washed cell 2 times, with 160 μ lPBS re-suspended cells, the in-vitro transcription product RNA(compared with control cells adding the above-mentioned purifying of 5-10 μ g adds the PBS of same volume), carry out electricity according to the GenepulserXcell of the experiment parameter Biorad of voltage 140V, burst length 25ms, pulse 1 time after mixing and turn.Then cell is placed in 6min on ice, then proceeds to 37 DEG C of cultivations in the MEM substratum containing 10% calf serum.Substratum is replaced by the MEM substratum containing 2% calf serum by next day, continues to be cultured to 5th ~ 7 days, and occur obvious CPE with the BHK21 cell that pJD4FC in-vitro transcription thing electricity turns, compared with control cells is without pathology (Figure 48).By sick cell and supernatant liquor multigelation 3 times, the centrifugal 10min of 4000rpm, results supernatant liquor, in-70 DEG C of preservations after packing, the first time embedded virus called after JD4(P1 of acquisition).

6, the Secondary Culture of encephalitis/Dengue 4 type embedded virus

The first-generation embedded virus JD40.5ml getting above-mentioned acquisition inoculates the primary hamster kidney cell (PHK cell) of adherent growth, after 37 DEG C of absorption 1h, adds the MEM substratum containing 2% calf serum, continues to be cultured to cell and occur CPE.Aspirate supernatant, the centrifugal 10min of 4000rpm, the supernatant liquor of results is s-generation embedded virus JD4(P2), in-70 DEG C of preservations after packing.By the same way JD4 was uploaded to for the 10th generation at PHK cell, every qualification (Figure 49) is carried out to embedded virus therebetween.

Two, the RT-PCR qualification of encephalitis/Dengue 4 type embedded virus

1, the RT-PCR of encephalitis/Dengue 4 type embedded virus identifies and order-checking

Extract s-generation embedded virus JD4(P2 with the HighpureviralRNAkit of Roche company) RNA, and with the Super of Invitrogen embedded virus RNA reverse transcription is cDNA by IIIReverseTranscriptase.With this cDNA for template, carry out pcr amplification with the PrimeSTARHSDNApolymerase of Takara.Primer sequence is as follows: first couple of JD4PF1:5'-CACCTGATCACGTCGGAATA-3', JD4PR1:5'-CCGCTCGAGCCCAAAGCTTCAAACTCTAGAT-3', product length 2078bp; Second pair: JD4PF2:5'-CGCCCCTACGATTCCGGACAGA-3', JD4PR2:5'-GGAAAAGGATCCGTGGTTCCAGG-3', product length 2155bp; 3rd is right: JD4PF3:5'-TGCGGAGTCAGATCTGTCACT-3', JD4PR3:5'-CATTTTCTGTCCGGAATCGTAG-3', product length 820bp; 4th is right: JD4PF4:5'-GAACATGGAGGATGCGTCAC-3', JD4PR4:5'-GGTCCGGACCAGTCTAGTGACAGATCTGACTC-3', product length 1608bp.Result shows 4 products and all increases successfully (Figure 50).PCR primer is checked order, all consistent with embedded virus expected sequence.

2, the plaque of encephalitis/Dengue 4 type embedded virus JD4 detects

BHK21 cell is seeded to six orifice plates, when cytogamy degree reaches 70-80%, carries out plaque detection.Using serum-free MEM substratum as diluent, respectively 10 times of gradient dilutions are carried out to each generation embedded virus.Suck the substratum of BHK21 cell, in each hole, add the virus liquid after 400 μ l dilutions respectively, in the soft jolting of 37 DEG C of absorption 60min, every 20min once.Suck virus liquid after having adsorbed, in every hole, add 2ml1% low melting-point agarose (containing 2% calf serum), in 37 DEG C of cultivations after to be solidified.5 days afterwards every hole add 2ml4% paraformaldehyde and fix 60min, then with the dyeing of Viola crystallina dye liquor, observe plaque form and also calculate virus titer (Figure 51) according to plaque quantity.

3, the drafting of encephalitis/Dengue 4 type embedded virus JD4 growth curve

With containing the MEM maintenance medium (Gibco) of 2% calf serum by JEV/D4(P3) to be diluted to infection multiplicity (MOI) be respectively 0.1,0.01,0.001 to virus liquid, the PHK cell of adherent growth in inoculation T75 bottle, in the soft jolting of 37 DEG C of absorption 60min, every 20min once.Suck virus liquid after having adsorbed, add the MEM maintenance medium (Gibco) of 20ml containing 2% calf serum, in 37 DEG C of cultivations.Extracted 0.5ml supernatant liquor every 12 hours ,-70 DEG C of preservations are to be measured, supply volume with 0.5mlMEM maintenance medium, and sample collecting is to inoculating latter 96 hours.Carry out titer determination according to each time point sample of plaque method to different MOI, draw the growth curve (Figure 52) of JD4 virus in PHK cell.Result display virus titer rises rapidly after inoculation, and 36-48h reaches the highest after inoculation, after this declines rapidly.The virus titer of high MOI inoculum size occurs that peak value comparatively early, but the peak value corresponding to different MOI inoculum size is without significant difference.

4, the Westernblot qualification of encephalitis/Dengue 4 type embedded virus JD4

At 80 μ l third generation embedded virus JD4(P3) in add 20 μ l5 × proteinloadingbuffer, boiling water boiling 10min after mixing, the centrifugal 1min of 10000rpm, get supernatant liquor and carry out SDS-PAGE electrophoresis, protein delivery is to nitrocellulose filter, close 1h with 5% skimmed milk room temperature, then use dengue virus 4 type E protein polyclonal blood antibody (extent of dilution 1:40 is prepared voluntarily) in 4 DEG C of overnight incubation.PBS washes film, then with goat anti-rabbit igg (extent of dilution 1:5000, the green skies, Shanghai) the incubated at room 1.5h of AP mark.Develop the color with BCIP/NBT alkaline phosphatase colouring reagents box (the green skies, Shanghai) after PBS washes film.According to the same terms, encephalitis b virus JEV (P3) is detected, in contrast.

Result display encephalitis b virus has no band, and embedded virus can be observed clear band at 55kD place, corresponding with dengue virus E protein position, result and expection consistent (Figure 53).

5, the immunogenicity experiments of encephalitis/Dengue 4 type embedded virus JD4

(1) immunogen preparation

Gather in the crops the third generation encephalitis/Dengue 4 type embedded virus supernatant cultivated in PHK cell, measure after virus titer through plaque method, with MEM basic medium (Gibco), virus titer is adjusted to 10 ⁵/ ml is for subsequent use.

(2) laboratory animal and grouping

60 Healthy female BALB/c mouse in 4 week age is provided by Chengdu Institute of Biological Products Co., Ltd..Laboratory animal is divided into 2 groups at random, often organizes 30: (1) JEV/D4 embedded virus immune group; (2) negative control group (MEM basic medium).

(3) immunization protocol

Get above-mentioned virus liquid and negative controls, fully pass through abdominal injection approach Mice Inoculated after mixing, 500ul/ only.After initial immunity, 2 weeks with identical approach and dosage booster shot once.Initial immunity starts blood sampling after 3 weeks, after this every 2 weeks 1 time to the 13rd week is terminated, each 5, amounts to 6 times.Separation of serum, 56 DEG C of deactivations 30 minutes after diluting 10 times with PBS ,-20 DEG C save backup.

(4) inspection of immune serum NAT

Above-mentioned serum sample (1:10) serum dilution (PBS) is carried out doubling dilution to 1:640 according to 1:2, every concentration samples is got 200ul respectively and is contained 50-100PFU dengue virus 4 type with same volume and mix, after hatching 1 hour in 37 DEG C after mixing, join and cover with on six orifice plates of BHK21 cell, measure NAT.Negative control is done not add sero-fast MEM substratum.Using can in and the serum-concentration of most high dilution of 1/2 virus as NAT.

Result shows, after encephalitis/Dengue 4 type embedded virus immune mouse, can produce the neutralizing antibody of special anti-homotype dengue virus, the highest NAT is that 1:22.36(is shown in Figure 54).

Description of test, the present invention successfully builds and obtains encephalitis/Dengue 4 type embedded virus, its can in PHK cell massive duplication, can produce the neutralizing antibody of dengue virus, immunogenicity is strong, can be prepared as dengue vaccine.

To sum up, the present invention successfully constructs infection and encephalitis B virus infection sex clone, and the Japanese encephalitis/dengue chimeric virus constructed further containing Dengue 1 type, Dengue 2 type, Dengue 3 type or 4-type dengue virus antigen gene prM-E, these embedded viruses can on various human production of vaccine cell growth and breeding, growth characteristics are similar, may be used for the dengue vaccine of production four serotypes, and, these four kinds of embedded virus immunogenicities are strong, the titre of the antibody produced is high, has good prospects for commercial application.

Claims (2)

1. embedded virus, is characterized in that: its nucleotide sequence is as shown in any one of SEQIDNO.8 ~ 11.

2. embedded virus according to claim 1 is preparing the purposes in dengue vaccine.