CN101712965A - Japanese encephalitis virus JEV replicon vector and application thereof - Google Patents

Abstract

The invention provides a replicon vector taking Japanese encephalitis virus genome as a framework, as well as a cell line for packaging the same and a packaging system. The JEV replicon vector has the capability of efficiently expressing foreign protein. Mice are immunized with the replicon vector, and the titer of an anti-JEV antibody reaches 1:1280 after three immunizations so as to protect 75 percent of suckling mice from virus attack. The cell line provided can produce 1.6*105 U/ml of pseudovirus particles after packaging JEV replicon, and the titer of the anti-JEV antibody reaches 1:2560 after two immunizations so as to protect 73 percent of suckling mice from virus attack. The invention establishes a technical platform for a JEV replicon vector system for the first time, and explores the feasibility of researching replicon vaccines and pseudovirus vaccines through the JEV replicon vector system, thereby laying a solid foundation for developing and researching a plurality of novel vaccines used to prevent and treat tumors and viral diseases in future.

Description

A kind of Japanese encephalitis virus JEV replicon vector and application thereof

Technical field

The present invention relates to a kind of is the replicon carrier of skeleton with the japanese encephalitis virus genome, and the clone and the packaging system that are used for this replicon carrier packing.More specifically, the present invention relates to a kind of can be as the JEV expression system of vaccine delivery system, immunogenic protein and polypeptide that this expression system can encoding exogenous.Vaccine can pass through DNA, the perhaps form of virus-like particle and using, utilize this carrier system with and derived products can be engaged in gene therapy, the research of gene prevention.

Background technology

JEV belongs to the flaviviridae yellow fever virus and belongs to, and by killing propagation, is human important pathogenic former, can cause permanent nervous system disease, or even fatal disease.Have every year to surpass 50,000 case, lethality rate surpasses 25%, and the survivor who surpasses half in addition has persistent nervosa sequela.JEV mainly is distributed in the Asia, and from the Soviet Union to India, China removes Xinjiang, and all there is morbidity in Tibet outside the Qinghai, still have several thousand routine patients now every year, and the people's health is caused great harm, is classified as Category B notifiable disease by the Ministry of Health.Along with the warming of global climate, this disease epidemic regions has also been found the propagation of JEV also in continuous expansion in the Southern Hemisphere in recent years, and JEV threatens to world's public health.

JEV is a little enveloped virus, genome is a sub-thread justice RNA chain, be approximately about 11KB, genome comprises a big reading frame, both sides are 5 ' and 3 ' non-coding region (NTRs), the both sides non-coding region is the important cis response element of genome duplication, the RNA chain of JEV has the cap sequence of an I type at 5 ' end, and 3 ' end lacks poly (A) tail, ORF can translate into a big polypeptide protein, in when translation and translation post-treatment is 3 structural protein and 7 Nonstructural Proteins, C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, at present owing to lack an effective reverse genetics system, to duplicate about protein function and the JEV of JEV, neurovirulent molecular mechanism information lacks very much.

The flavivirus genome has a remarkable characteristic, its RNA has the self-replicating ability, has infectivity simultaneously, after naked RNA enters sensitive cells, and self-replicating in a large number, and further translate into corresponding viral protein, become complete virion in born of the same parents' internal packing.The yellow fever virus replicon is often referred to the structure gene on the genome is partly cut down, and keep complete nonstructural protein gene, subgenomic RNA has kept the self-replicating ability like this, it not only can express Nonstructural Protein, and can express the structural protein of reservation and/or the foreign gene of interpolation.Theoretically, this subgenomic replication is equal to complete genome, and in reproduction process, positive chain RNA is exponential increase.If in this replicon, insert foreign gene, along with replicon rna amplification in a large number in cell cytosol, just make that foreign gene obtains effective expression, therefore, replicon rna can be used as the good carrier of expression alien gene.

Because rna replicon can self-replicating, and ability with inside and outside transgenosis, can be in the part instantaneous high level expression foreign gene, at present, be used to the development of the vaccine of antiviral vaccine and anticancer disease based on the positive chain RNA virus carrier of replicon, and field such as oriented carrier preparation.In addition, rna replicon also is a kind of useful instrument, can be applicable to explore the research of aspects such as virus replication, mechanism of causing a disease and antiviral screening.Yellow fever virus rna replicon comprises the cis-acting elements of viral genome 5 ' end and 3 ' end non-coding region, whole Nonstructural Protein (duplicating/transcriptase) gene, and structural protein gene disappearance or alternative by foreign gene.

If replicon carrier is aided with the assistant carrier cotransfection cell of expressing virus structural protein, can repack into new pseudovirion.Theoretically, this pseudovirion only has " single infection ability ", because the geneome RNA of its packing is a replicon of having removed structure gene, virion again behind the cells infected owing to lack the structural protein of virus, can not repack becomes virion, can not infect peripheral cell.The pseudovirion vaccine is removed because of the portion gene of carrier, and the capacity that can insert gene fragment increases, and pseudovirus can only single infection, can't breed after entering in the body, so security and controllability are higher.Pseudovirus Packaging Method commonly used at present is to provide packaging structure albumen by helper virus, but this packing causes the generation of recombinant virus easily, causes potential safety hazard.And pseudovirus can not go down to posterity in non-packing cell, so the precondition that structure can be stablized, the clone of packaging virus has become pseudovirus vaccine application development efficiently.Pseudovirion based on replicon does not have infectivity, and can not integrate with cellular genome, but can induce general immunity, mucosal immunity and the reaction of MHC-1 Restricted CTL, and the not interference of the interior existing carrier antibody of acceptor.These characteristics make it can be used as a kind of transgenosis platform, can be used for developing the preventative and therapeutic vaccine of transmissible disease and tumour.Developed at present some vaccine and tumor treatment and preventative vaccines, and on a lot of disease models, achieved success based on replicon.

The sub-RNA of virus replication can send by three kinds of modes: 1) the naked RNA (based on RNA) of in-vitro transcription, the cDNA of rna replicon is placed SP6 or the control of T7 promotor down, utilize SP6 or the T7 polymerase of phage to carry out in-vitro transcription, RNA direct transfection cell opens the beginning rna replicon, realizes the high level expression of foreign gene; 2) be built into plasmid DNA, it is the DNA/RNA carrier, introduce II type rna polymerase promoter in replicon cDNA upstream to start transcribing of replicon cDNA, by being transcribed into replicon rna (based on DNA) in the cell RNA polymerase II body, behind the dna vector transfectional cell, fs: the rna plymerase ii promotor is initial synthetic RNA in nuclear, is transported in the tenuigenin through processing with after modifying, and is equivalent to replicon rna; Subordinate phase: this RNA self-replacation and expression alien gene, be equivalent to the viral RNA replication cycle, thereby replicon is realized the high level expression of foreign gene.3) provide structural protein that replicon rna is packaged into pseudovirion (PIPs) by trans-complementation system (assistant carrier or clone), and Nonstructural Protein and structural protein encoding gene lay respectively on expression vector and the assistant carrier, and assistant carrier does not have packaging signal, can not be packaged, so behind the pseudovirion cells infected of preparation, only can carry out taking turns duplicating, and irreproducible filial generation no longer has infectivity.

The virus host cell of Flavivirus is extensive, and general susceptible has comprised various kinds of cell types such as insect, birds, Mammals, and flavivirus all can infect in these host cells, duplicate.Simultaneously, go down to posterity, produce the virus of high titre but simple relatively genome structure and reproduction process make virus repeatedly duplicate within a short period of time.In addition, viral RNA is finished in tenuigenin and duplicated amplification, has eliminated to enter nuclear possibility, can not be incorporated in the cellular genome, and is comparatively safe.Surplus nearest ten year, use the flavivirus carrier and carry out the existing many reports of expression of exogenous gene: as, DENV, YFV, WNV, KUNV, TBEV etc.(reference 12-16)

Elder brother Tianjin virus KUNV replicon is the yellow fever virus replicon that successfully constructs and be used to express exogenous antigen at first, and usefulness KUN virus replication such as Varnavski have been expressed CAT, GFP, β-gal (reporter gene) and HCV-C (160 and 191 residue), HCV-NS3, VSV-G respectively.Anraku etc. express the many epitopes of mouse by sending with 3 kinds of different KUN virus replication; can induce protectiveness CD8+T cell immune response, by these 3 kinds of differences send mode once immunity just can induce and express 3 suitable CD8 of immunity of same immunogenic vaccinia virus recombinant ⁺T cell response, only 0.1 μ g just can induce and the suitable CD8 of the conventional dna vaccination of 100 μ g based on KUN virus replication of DNA ⁺T cell response behind naked RNA (replicon rna) immune mouse, produces the provide protection at vaccinia virus recombinant and B16 tumour cell, and these results show the important value of replicon carrier in inducing anti-disease poison and antitumor provide protection.Harvey etc. study the HIV vaccine with the Gag antigen protein that 3 kinds of KUN virus replication subsystems are expressed HIV-1, and immune mouse can be induced the specific CD8 of tangible anti-Gag ⁺T cell response is compared with vaccinia virus recombinant, can induce the CD8 of generation with the pseudovirus initial immunity ⁺Nearly 4.5 times of T responsive cell numbers, booster immunization can provide tangible provide protection at vaccinia virus recombinant; Even more important result is CD8 after the PIPs immunity ⁺The T cell can be secreted gamma-interferon for a long time, makes this provide protection reach the 6-10 month.This result shows that KUN virus replication subsystem provides the carrier that potential value is arranged for the HIV vaccine design.

Other are such as WNV, YFV, TBEV, the replicon carrier system of viruses such as DEN-4 is also well developed, in this research, we wish the strain based on JEV SA14-14-2, make up JEV sub-genome duplication, and further set up JEV replicon carrier system, its exploitation is become the new platform of vaccine research.

Summary of the invention

First purpose of the present invention provides a kind of JEV replicon carrier.

Second purpose of the present invention provides a kind of and is used for the clone that this replicon carrier is packed.

The 3rd purpose of the present invention provides a kind of JEV pseudovirus packaging system.

The 4th purpose of the present invention provides a kind of pseudovirion.

The 5th purpose of the present invention provides the method for the above-mentioned pseudovirion of preparation.

Last purpose of the present invention provides the application of above-mentioned JEV replicon carrier, clone, packaging system and pseudovirion.

Aspect first, provide a kind of JEV replicon carrier of the present invention, it is to be framework construction with the japanese encephalitis virus genome, whole preM genes and the part E gene of described replicon carrier disappearance JEV.Part E genetically deficient described here is meant that E albumen can not express or loss of activity.

Above-mentioned replicon carrier comprises the following element that can be operatively connected: promoter sequence; 5 ' end non-coding region; The C protein-coding region; Multiple clone site; The NS1 signal peptide coding region; The Nonstructural Protein coding region; 3 ' end non-coding region.

Above-mentioned promotor can be CMV promotor or T7 promotor.

Preferably 3 ' end in described multiple clone site has 3 ' end releasing member, perhaps has 5 ' end releasing member before described NS1 protein signal peptide-coding region.Described releasing member is preferably the nucleotide sequence of coding foot and mouth disease virus self lytic enzyme.

In an example of the present invention, described japanese encephalitis virus is the SA14-14-2 virus strain, makes up the nucleotide sequence of the replicon carrier that obtains shown in SEQ IDNO.22 by this virus strain.Certainly, those skilled in the art can be under the prerequisite that does not influence the replicon carrier function, described replicon carrier is modified, for example, replace, increase or reduce one or more Nucleotide at non-coding region, do not influencing under the active prerequisite of proteins encoded in the coding region, the amino acid whose codon of similar performance is being replaced, perhaps selecting other codons according to host's preferences according to the degeneracy of codon.

According to above-mentioned replicon carrier, those skilled in the art can with foreign gene particularly the structural protein encoding gene be inserted in the multiple clone site of described replicon carrier, obtain recombinant vectors.For this reason, the present invention also provides a kind of recombinant vectors that is obtained by JEV replicon carrier and foreign gene reorganization.Described foreign gene can be the gene of coding reporter protein or the gene of coding immunogenic protein.This immunogenic protein can be any immunogenic protein that can express in eukaryotic cell.

In an example of the present invention, described recombinant vectors comprises following structure:

1) a kind of coding can not produce the nucleotide sequence of the JEV replicon of infectious virus, wherein said JEV replicon carrier whole JEV Nonstructural Proteins of encoding.

2) at least one extraneous nucleotide sequence.Exogenous nucleic acid can be encoded by the pathogenic organisms body, for example virus, protein that fungi, bacterium, protozoon, invertebrates such as parasite and arthropods produced or that obtain, perhaps can encode by the sudden change that animal produced or that obtain, carcinogenic or tumprigenicity protein such as the tumour antigen that comprise the animal and human, exogenous nucleic acid is codified synthetic or artificial proteins also, as the immunogenicity epi-position that is used for induction of immunity that makes up.

3) can handle the promotor CMV/T7 of the nucleotide sequence of JEV replicon carrier delivery form.

4) nucleotide sequence coded a kind of foot and mouth disease virus 2A oneself protease of coding oneself protease.

Can in the host, pack in order to make above-mentioned replicon carrier or expression vector, the present invention also uses a kind of carrier for expression of eukaryon of expressing the japanese encephalitis virus structural protein, its can with above-mentioned JEV replicon carrier or recombinant vectors cotransfection host, thereby be packaged to be the JEV pseudovirion.The expression product of described carrier for expression of eukaryon comprises preM albumen and E albumen at least.Certainly, those skilled in the art also can express these two albumen respectively with different expression vectors.

In an example of the present invention, described carrier for expression of eukaryon is pCJECME.

Further the present invention also provides a kind of clone, and this clone contains above-mentioned carrier for expression of eukaryon.In an example of the present invention, described clone is the BHK-21 cells of pCJECME stable transfection.

On the basis of the above, the present invention also further provides a kind of JEV packaging system, and it comprises: 1) JEV replicon carrier recited above or recombinant vectors; And 2) clone recited above.

The present invention also provides a kind of JEV pseudovirion, and it is to be obtained by above-mentioned JEV packaging system packing.

The present invention further provides the method for the above-mentioned JEV pseudovirion of preparation, it is that above-mentioned JEV replicon carrier or recombinant vectors are imported in the described clone through the packing acquisition.

Particularly, above-mentioned pseudovirion can be obtained by following steps:

1) above-mentioned carrier for expression of eukaryon is imported in the BHK-21 cell, screen the cell clone of high expression level JEV structural protein, thereby further screen the package cell line of stably express JEV structural protein through G418.When described packing cell can stably express JEV structural protein, the amplification culture packing cell.

2) import above-mentioned one or more JEV replicon carrier or recombinant vectorss to described packing cell, cultivated 5-7 days.

3) reclaim, purifying contains the genomic pseudovirion of JEV replicon.

The present invention also provides above-mentioned JEV replicon carrier or recombinant vectors, above-mentioned carrier for expression of eukaryon, above-mentioned clone or the application of above-mentioned JEV packaging system in preparation amplicon dna vaccine.

The present invention also provides a kind of vaccine that contains above-mentioned JEV pseudovirion, is used to carry out the research of gene therapy or gene prevention.

JEV replicon carrier of the present invention has the ability that efficiently expresses foreign protein.With the replicon carrier immune mouse, three exempt from the anti-JEV antibody titers in back reaches 1: 1280, can protect 75% suckling mouse to avoid virus attack.The clone that provides can produce 1.6 * 10 behind the packing JEV replicon ⁵The pseudovirion of U/ml, two exempt from mouse after anti-JEV antibody titers reach 1: 2560, can protect 73% suckling mouse to avoid virus attack.The present invention has set up the technology platform of JEV replicon carrier system first, and explored the feasibility that JEV replicon carrier system carries out amplicon dna vaccine and pseudovirus vaccine research, the new generation vaccine that is used to prevent and treat tumour and virus disease for development research from now on is multiple has been established solid basis.

Description of drawings

What Fig. 1 showed is that indirect immunofluorescence (IFA) detects the expression of JEV replicon carrier pCMW-2M in the BHK-21 cell; Wherein, the IFA result of A:pCMW-2M transfection BHK-21 cell after 3 days; B: the negative contrast of BHK-21 cell of untransfected.

Behind JEV replicon pCMW-2M usefulness Xba I linearization for enzyme restriction, scabble with MBN again, after fragment reclaims purifying, liposome transfection BHK-21 cell.The BHK-21 cell of getting after the transfection 3 days carries out IFA, analyzes the expression of replicon carrier pCMW-2M in the BHK-21 cell, does one anti-ly with anti-JEV cavy polyclonal antibody, does two anti-ly with the anti-cavy IgG of the goat of flag F ITC, and Azo-Blue dyes.30% the cell of having an appointment shows as positive findings, presents green fluorescence, and endochylema is expressed.And negative control group is observed under fluorescent microscope and is rendered as garnet.

What Fig. 2 showed is the expression of different time green fluorescent protein behind the pCMW-2M-EGFP transfection BHK-21 cell; Wherein, A: 24hr after the transfection; B: 72hr after the transfection; C: 7d after the transfection; The BHK-21 cell of the pCMW-2M-EGFP defective type mutant plasmid transfection of D:ORF frameshit is cell in contrast.

With plasmid pCMW-2M-EGFP with Xba I linearizing after with liposome transfection BHK-21 monolayer cell, the defective type replicon carrier pCMW-2M-EGFP that sets up a hole transfection phase shift mutation simultaneously is as negative control.Observe every day after the transfection.As can be seen from Figure 2, can observe the proteic expression of EGFP in transfection in microscopically after 24 hours, positive cell on the 3rd increases after the transfection, and fluorescent signal strengthens to some extent.After transfection the 7th day, positive cell can reach 20%, and can obviously observe the phenomenon that the prolongation positive cell along with the transfection time increases down in mirror.

What Fig. 3 showed is the expression of reorganization JEV replicon pCMW-2M-LAC in suckling mouse brain that contains the LacZ reporter gene; Wherein, A: replicon carrier pCMW-2M-LAC injection mouse is original position dyeing testing goal expression of gene after 4 days; B:pCMV-Lac injection mouse original position coloration result after 4 days; The C:PBS contrast.

In order further to understand JEV replicon carrier expression in animal body, the recombinant plasmid vector pCMW-2M-LAC that makes up is detected evaluation in the ability of mouse expression in vivo LacZ.Choose 3-4 age in days Kunming kind suckling mouse, respectively intracerebral injection 20 μ l (5 μ g) plasmid pCMW-2M-LAC, pCMV-Lac or PBS.After 4 days, dissect suckling mouse, by the expression of X-Gal original position staining examining report gene in cerebral tissue, can observe directly large stretch of tissue at the cerebral tissue position of replicon carrier pCMW-2M-LAC injection and be blue, the cerebral tissue position of conventional dna vector pCMV-Lac injection only is dispersed in the point-like blueness, and the cerebral tissue position of PBS group injection does not observe Bluepoint.

What Fig. 4 showed is the measurement result of the anti-JEV antibody titers of serum behind the replicon carrier immune mouse;

With the JEV replicon pCMW-2M of 50 μ g and pCMW-G2-R with the dna form immune mouse, get blood from mouse tail vein after two weeks, detect the anti-JEV antibody titers of serum with indirect ELISA, along with increasing of immune time, the anti-JEV antibody titers of serum progressively increases, the anti-JEV antibody titers of replicon pCMW-2M immune mouse inductive is a little more than pCMW-G2-R immune mouse inductive antibody titers, and both differ not significantly (P＞0.05).PCMW-2M immune mouse three is exempted from the anti-JEV antibody titers in back and is reached 1: 1280, and replicon carrier group and PBS group and carrier pCMW-118L group antibody titers differ significantly (P＜0.05).

What Fig. 5 showed is the measurement result of the anti-JEV NAT of serum behind the replicon carrier immune mouse;

Get the mice serum in two weeks of back of immunity for the third time, reduce the titre that serum JEV neutralizing antibody is determined in experiment by 50% plaque.As can be seen replicon pCMW-2M for the third time the anti-JEV NAT of immune serum reach 1: 32, a little more than pCMW-G2-R immune mouse inductive NAT, both differ not significantly (P＞0.05).But two replicon carrier groups and PBS group and carrier pCMW-118L group NAT differ significantly (P＜0.05).

What Fig. 6 showed is that Western Blot detects the proteic expression of E among the cell strain CME-4; Wherein, the 1. cell pyrolysis liquid of cell clone CME-4; 2. normal BHK-21 cell pyrolysis liquid; M. be Marker.

With cell clone CME-4 and the cracking respectively of normal BHK-21 cell, detect the former cell pyrolysis liquid by WesternBlot the proteic expression of E about 53kD is arranged, conform to the expection size, and no specific band manifests in the contrast, shows the expression that JEV E structural protein are arranged in the clone;

What Fig. 7 showed is the genomic PCR qualification result of positive cell clone; Wherein, 1,2,3: positive cell clone; The contrast of 4:BHK-21 cell; M:DNA Marker IV.

Extract primary dcreening operation and express the genome of JEV structural protein male monoclonal cell, with pJEVc1 (SEQ ID NO:16)/dXhoE (SEQ ID NO:17) is primer, the genome product is a template, the total length 2396bp fragment of amplification JEV C-prM-E gene, extract normal BHK-21 cellular genome in addition as negative control, electrophoresis showed is successfully amplified the segmental cell clone of purpose pick out.As can be seen, amplification has obtained the specific fragment of JEV in the positive cell genome among Fig. 7, and size and expection are consistent, and negative control group fails to amplify the expection band.The result shows that the C-prM-E gene has been integrated in the genome of BHK-21 cell.

What Fig. 8 showed is that IFA detects clone CME-4 stably express JEV structural protein situation; Wherein, A, B are the proteic clone of expression structure after 2 strains were gone down to posterity 5 months; C is a BHK-21 cell negative control.

CME-4 clone cultured continuously 5 months, go down to posterity more than 100 times after, through indirect immunofluorescence, with the anti-JEV of the cavy anti-proteic situation of positive cell CME-4 expression structure of identifying how.As can be seen, the ratio of the proteic cell of expression structure still accounts for more than 50% in the clone that screening obtains, and the result is consistent with initial test.Illustrate that clone that we obtain can be stablized and the continuous expression structural protein, obtained to be used to preferably prepare the package cell line of JEV pseudovirion, for the acquisition of follow-up pseudovirion is had laid a good foundation.

What Fig. 9 showed is the egfp expression of different time points behind pCMW-2M-EGFP transfection BHK-21 cell and the CME-4 cell.Wherein, ABC shows that pCMW-2M-EGFP transfection BHK-21 cell DEF shows pCMW-2M-EGFP transfection CME-4 clone A, 24 hours B after the D transfection, 72 hours C after the E transfection, after the F transfection 120 hours.

After will comprising the JEV replicon carrier pCMW-2M-EGFP transfection structural protein clone CME-4 of reporter gene, the expression amount of reporter gene has had significant raising, the different time points of replicon carrier pCMW-2M-EGFP behind transfection BHK-21 cell, the expression amount of EGFP increases along with the prolongation of time, reaches as high as about 20%.But behind transfection package cell line CME-4, the expression amount of EGFP improves, and the cell of expressing EGFP in transfection after 24 hours accounts for about 20%, and positive cell increases to about 30% after 72 hours, and fluorescent signal is stable, and endochylema is expressed.Transfection still had the positive cell rate of 30-40% in 120 hours later on.

What Figure 10 showed is after replicon carrier pCMW-2M-LAC distinguishes transfection package cell line CME-4, the expression of LacZ; Wherein, A.pCMW-2M-LAC transfection CME-17 clone; B.pCMW-2M-LAC transfection CME-4 clone; C.pCMW-2M-LAC transfection BHK-21 cell.

Behind the replicon carrier transfection packing cell, expression amount is significantly improved, and after linearization of the pCMW-2M-LAC plasmid DNA transfection structural protein clone of 1.6 μ g, the cell expressing LacZ about 20% is arranged in 12 orifice plates, and cell indigo plant is dyed obviously, and the prompting expression amount is higher.This has proved that also the JEV replicon carrier has good replication, and clone has capacity packing;

That Figure 11 shows is replicon carrier pCMW-2M-LAC transfection package cell line CME-4, and the reporter gene expression situation after the amplification culture; Wherein, A: the culture supernatant behind the replicon carrier pCMW-2MLac transfectional cell series CME-4 is cells infected system once more; B: the double-dyed clone CME-4 C of sense of continuity: the clone CME-4 that consecutive infection is three times; D: the culture supernatant behind the replicon carrier pCMW-2MLac transfectional cell series CME-4 infects normal BHK-21 cell.

Culture supernatant is continued cells infected system, and the expression that detects Lac Z after 4 days has the cell expressing Lac Z about 50% as can be seen approximately, and sees that from the dyeing situation expression amount is suitable with the first-generation shown in A in the s-generation clone.Gathering in the crops this expression supernatant continuation cells infected is CME-4, and the expression of Lac Z is shown in B.Positive cell number is more than the s-generation as can be seen, and the equal indigo plant of most of cell is dyed, and relative expression amount is weaker than s-generation cell.After continuing to infect for the third time, the ratio of positive cell descends more obvious, shows that as figure C expression amount also descends.After first-generation culture supernatant infected normal BHK-21 cells, positive rate did not further improve, and expression amount is lower, shown in figure D.

Figure 12 is the Electronic Speculum figure of JEV pseudovirion negative staining; A wherein: magnification 93000 * B: magnification 135000 *.

In the cell conditioned medium of pseudovirion, negative staining all can be observed virion, and is how rounded, and diameter between 40-60nm, is consistent with the structure of JEV greatly.And the hollow shape that exists minority to be dispersed in do not have nuclear particle, about the about 20nm of diameter, analyzes the virus-like particle (VLPs) that constitutes into prM-E, owing to do not contain the nucleocapsid structure, so be cavity shape globosity, big or smallly conforms to bibliographical information.

Figure 13 is the RT-PCR electrophoresis result; Wherein, 2,3: pseudovirus RT-PCR product (810bp); 6,7: pseudovirus RT-PCR product (164bp); 1: negative control 5:BHK-21 cell contrast 4,8:DNA Marker III.

Behind the pseudovirion infection BHK-21 cell with results, with metainfective cell harvesting, extract RNA, obtain cDNA first chain by reverse transcription, with primer CX12 (SEQID NO:18)/P3R (SEQ ID NO:19), CX16a (SEQ ID the NO:20)/genome 7352-8171 sequence of CX16b (SEQ ID NO:21) amplification pseudovirion and the specific fragment of 9316-9479nt sequence, length is respectively 810bp, 164bp;

Figure 14 is an antibody titers level view behind JEV pseudovirion PIPs, the package cell line culture supernatant immune mouse;

Behind the JEV PIPs immune mouse, can produce proteic specific antibody reaction at E, antibody titers reaches 1: 2560 after immune 2 times, in the CME-4 cell conditioned medium because of containing the E albumen of expression, so also can produce specific at the proteic antibody response of E, the immunity 2 times after antibody titers reach 1: 640; See Figure 14, antibody horizontal and PBS control group significant difference (P＜0.05) in JEV pseudovirus immune group and the package cell line culture supernatant immune group serum;

Embodiment

The present invention has disclosed a kind of replicon carrier by JEV, the JEV replicon carrier system that the clone of derivative vector and expression JEV structural protein makes up.JEV replicon carrier of the present invention can long-acting expression foreign protein, is used for the development of replicon carrier vaccines; JEV stabilising packaging clone among the present invention, C, prM and E albumen that can stably express JEV, this clone can realize packing the JEV replicon carrier and form pseudovirion, and can form the pseudovirion of higher titre after the optimal conditions, further simplifies production stage.

Further specify content of the present invention below in conjunction with figure and embodiment, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.

If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.

The preparation process of embodiment 1, replicon carrier pCMW-2M

1. the transformation of plasmid vector pMW118 (Nippon Gene)

With low copy pMW118 (Nippon Gene) plasmid vector is that transform on the basis, at first design two oligonucleotide sequence D118 (SEQ ID NO:2) and P118 (SEQ ID NO:3) that contain SalI-SacI-BamHI-MluI-KpnI-XbaI-EcoRI multiple clone site (MCS), be used to make up an adapter, be cloned among the plasmid vector pMW118 construction recombination plasmid pMW-118L by Sal I and EcoR I restriction enzyme site.

With plasmid pcDNA3.1 is template, goes out long be the CMV sequence of 607bp (CMV promoter sees SEQ ID NO:1) with primer pPCMV (SEQ ID NO:4) and primer pSCMV (SEQ ID NO:5) by pcr amplification.Be cloned among the plasmid vector pMW-118L by SphI and SalI restriction enzyme site, make up plasmid pCMW-118L.

2. the JEV replicon carrier pCMW-2M of JEV virus structural protein has been removed in preparation.

On the full-length infectious clone's of JEV vaccine strain SA14-14-2 basis, preparation JEV replicon carrier, described JEV vaccine strain SA14-14-2 is from Chinese medicine biological products assay institute.

With the full-length infectious clone of JEV pBR-JTF is template, goes out the fragment of long 557bp by pcr amplification with primer pT1L (SEQ IDNO:6) and primer dXFMDV (SEQ ID NO:7).With pBR-JTF is template, goes out to be about the fragment of 1515bp by pcr amplification with primer pX2MUN (SEQ ID NO:8) and primer dxMUN (SEQ ID NO:9).Bsa I enzyme is cut two PCR products, connects the back fragment purification.With this connector is template, primer pT1L/dxMUN about 2054bp fragment that increases.Cut rear clone in pBR-JTF by Sal I/Mfe I enzyme, construction recombination plasmid pBR-2M further is connected among the plasmid vector pCMW-118L by the replicon fragment of Sal I/Xba I double digestion with pBR-2M, makes up pCMW-2M.Same method has made up replicon carrier pCMW-G2-R, and both differences are the reservation figure place of E gene, and pCMW-2M keeps terminal 21 amino acid of E gene, and pCMW-G2-R has kept 30 amino acid of E gene end.

3. preparation contains reporter gene EGFP, the JEV replicon carrier of LACZ.

With plasmid pIRES2-EGFP is template, pass through pcr amplification EGFP gene order (SEQID NO:12) with primer pXEPL (SEQ ID NO:10) and primer doEGFP (SEQ ID NO:11), by Xho I and BsrG I restriction enzyme site reporter gene EGFP is inserted JEV replicon pCMW-2M, obtain recombinant plasmid pCMW-2M-EGFP.

With plasmid pCMV-Lac is template, pass through pcr amplification LAC Z gene order (SEQ IDNO:15) with primer plac (SEQ ID NO:13) and primer dlac (SEQ ID NO:14), by Xho I and BsrG I restriction enzyme site reporter gene LAC Z gene is inserted among the JEV replicon pCMW-2M, obtain recombinant plasmid pCMW-2M-LAC.

The Function Identification of embodiment 2, replicon carrier pCMW-2M

1. the Function Identification on the cell levels

Get 5 μ g left and right sides JEV replicon DNA as template, XbaI enzyme list is cut plasmid and is carried out linearizing, and MBN scabbles, behind the fragment purification.Liposome (LipofectamineTM 2000, Invitrogen company) method transfection BHK-21 cell.After the transfection 3 days, carry out indirect immunofluorescence experiment (IFA), analyze the expression of replicon in the BHK-21 cell, do with the anti-JEV polyclonal antibody in cavy source one anti-, do with the anti-cavy IgG of FITC labelled goat two anti-, Azo-Blue dyeing.See Fig. 1,30% the cell of having an appointment shows as positive findings, presents green fluorescence, and endochylema is expressed.And negative control group is observed under fluorescent microscope and is rendered as garnet.

2 expression of replicon carrier pCMW-2M-EGFP in the BHK-21 cell

Ability for the sub-vector expression foreign gene of checking copying, after being inserted with the replicon plasmid pCMW-2M-EGFP usefulness Xba I linearization for enzyme restriction of reporter gene EGFP, scabble with MBN, after fragment reclaims purifying, liposome transfection BHK-21 cell is at the different time expression of fluorescence microscope reporter gene EGFP in cell.See Fig. 2,24hr begins the positive cell that has obvious EGFP to express after the visible transfection, 3d after the transfection, and positive rate is up to 10%, and fluorescence intensity constantly strengthens, 7d 20% positive cell of having an appointment to the transfection, control wells is then negative all the time.

When making up pCMW-2M-EGFP, obtained an enzyme and cut and identify correct but order-checking finds that the other parts sequence is correct, thereby only in the NS1 encoding sequence, had a nucleotide deletion to cause the recombinant plasmid of ORF phase shift mutation.Get this recombinant plasmid transfection BHK-21 cell as negative control, the result does not observe green fluorescence all the time under fluorescent microscope.

3 expression of replicon carrier pCMW-2M-LAC in the BHK-21 cell

With being inserted with after the replicon carrier pCMW-2M-LAC linearizing of reporter gene LAC Z transfection BHK-21 cell respectively, set up a hole not make the normal BHK-21 cell of transfection simultaneously as negative control.Transfection is X-Gal original position dyeing detectionof after 4 days.Can observe the expression that LacZ is arranged behind the replicon carrier transfectional cell, but positive cell is less, expression amount heterogeneity, repeated experiments show that positive rate can not surpass 10%.

4 contain the expression of reorganization JEV replicon pCMW-2M-LAC in suckling mouse brain of LacZ gene

Choose 3-4 age in days Kunming kind suckling mouse, behind the extraction plasmid pCMW-2M-LAC, adjusting concentration with PBS dilution back is 250ng/ μ l, injects 20 μ l (5 μ g) plasmid pCMW-2M-LAC, pCMV-Lac or PBS in suckling mouse brain respectively.After 4 days, dissect suckling mouse, its cerebral tissue is taken out,, see Fig. 3 by the expression of X-Gal original position staining examining report gene in cerebral tissue.Coloration result shows, can observe directly large stretch of tissue at the cerebral tissue position of replicon carrier injection and be blue, and the cerebral tissue position of conventional dna vector pCMV-Lac injection only is dispersed in the point-like blueness, and the cerebral tissue position of PBS group injection does not observe Bluepoint.The result shows that the amplicon dna carrier is the high level expression reporter gene in vivo, compares with cytologic experiment, and positive signal is stronger, and area coverage is wide.

Embodiment 3, the experiment of replicon carrier pCMW-2M immune mouse

The grouping of 1 experiment mice:

Choose 3 age in week BALB/C mice, female, test, 6 every group, two groups of respectively immune replicon carrier pCMW-2M and pCMW-G2-R establish PBS group and empty carrier control group simultaneously.Concrete experiment grouping sees Table 1.

Table 1 replicon plasmid immune mouse experiment grouping sheet

2 immunization routes, injected dose:

Intramuscular injection i.m.: two thighs, inject the plasmid of 25 μ g in every leg quadriceps muscle of thigh through the PBS dilution.Every mouse needs immune ultrapure plasmid 50 μ g.

After the injection, immediately the injection site is carried out electricity irritation, WJ-2002 live body gene introducing apparatus parameter is: 100V, 50ms, after 6 pulses, turn polarity and carry out 6 pulses again.

3 immune times:

Interval two all booster immunizations, totally 3 times, immunizing dose is identical with initial immunity once more.

4 get blood opportunity and amount for taking blood:

Get blood from mouse tail vein, P0: get blood before the immunity; P1: 2 weeks behind the initial immunity; P2: 2 weeks of back of immunity for the second time; P3: 2 weeks of back of immunity for the third time.Amount for taking blood is about 100 μ L, 4 ℃, the centrifugal 20min of 5000g, separation of serum.Short term detection can place 4 ℃ behind the separation of serum, and long-term storage places-20 ℃.

The detection of anti-JEV specific antibody and neutralizing antibody in the 5 immune mouse bodies:

Reduce experiment detection specificity antibody and neutrality antibody by ELISA and 50% plaque.

(1) indirect ELISA

The preparation of ELISA envelope antigen:

Reorganization JEV live virus intracranial inoculation BALB/c mouse after the morbidity in 6 days, is collected the mouse brain, and-70 ℃ of refrigerators are placed, and take out multigelation 3 times during use, grind, and are dissolved in the complete cell culture medium of 2mL.4 ℃ of homogenate, 10, the centrifugal 10min of 000g.Protamine sulfate is added to supernatant, and to make ultimate density be 0.9% (mass/volume).Behind the incubated at room 2hr, 10, the centrifugal 10min of 000g, the supernatant that has comprised virion is used as the antigen that carries out ELISA.

The concise and to the point step of ELISA:

On 96 hole polystyrene plates, carry out ELISA, be used to detect antibody titers.Every hole 100 μ L carbonate-bicarbonate buffer (45.3mM NaHCO ₃, 18.2mM Na ₂CO ₃[pH9.6]) the middle 10 μ L virus liquid that adds, 4 ℃ of bags are spent the night.Second day, sealing, 1%BSA (comprises 0.1%Tween20) in PBST, 37 ℃ of sealing 2hr.Wash 3 times, every then hole adds the mouse serum of 100 μ L dilution, hatches 1hr for 37 ℃, washes 3 times.Every hole adds the anti-mouse IgG of HRP labelled goat of 100 μ L dilution, hatches 1hr for 37 ℃, washes 3 times.Add 100 μ L/ holes fresh preparation substrate solution [100mL phosphoric acid salt-citrate buffer solution (0.05mol/LNa ₂HPO ₄, the 0.24mol/L citric acid, pH 5.0), 40mg O-Phenylene Diamine (OPD), 15 μ L30%H ₂O ₂] colour developing 10～15min.The H that adds 50 μ L/ hole 2M ₂SO ₄Termination reaction.492nm and 630nm dual wavelength detect absorbancy.End dilution ELISA method is measured the specific antibody titre in the immune serum eventually.With the negative contrast of control group mice serum (being N), the OD value of dna vaccination immune group (being P) reaches more than 0.2, and P/N 〉=2.1 are positive.Every group average antibody titre is with antibody titers (geometrical mean ± standard deviation) expression in every group of single serum sample or a plurality of multiple holes of pooled serum.

The results are shown in Figure 4.As can be seen from the figure, along with increasing of immune time, the anti-JEV antibody titers of serum progressively increases, and pCMW-2M immune mouse three is exempted from the anti-JEV antibody titers in back and reached 1: 1280, and replicon carrier group and PBS group and carrier pCMW-118L group antibody titers differ significantly (P＜0.05).

(2) 50% plaque that detects the mouse NAT reduces test

Each immune group mouse is got each mice serum equal-volume respectively from every group to be mixed, 56 ℃ of deactivation 30min, the initial doubling dilution from 1: 4, with virus dilution (about 200PFU/ hole) balanced mix, virus after will diluting simultaneously and normal mouse serum balanced mix, in contrast, put 37 ℃ of water-bath 60min.

Adding respectively to cultivate in advance has in 24 well culture plates of BHK-21 cell, discards mixed solution after putting 37 ℃ of absorption 1hr, and every hole adds 1mL methylcellulose gum mixture continues to cultivate, day by day the observation of cell pathology.Each dilution sample standard deviation is established 3 multiple hole experiments, observation of cell infective virus situation behind the 7d.Discard the methylcellulose gum coverture, add crystal violet solution, 37 ℃ of dyeing 20min.Wash away crystal violet solution with flowing water, calculate the plaque number, the high dilution of the serum that reduces with 50% plaque is as NAT.

The results are shown in Figure 5, as can be seen replicon pCMW-2M for the third time the anti-JEV NAT of immune serum reach 1: 32 and PBS group and carrier pCMW-118L group NAT differ significantly (P＜0.05).

The detection of 6 cellular immunizatioies:

The mouse in 3 weeks is got spleen after the last immunity, carries out the mensuration of stimulation index (SI).

(1) preparation of splenocyte suspension:

The disconnected neck of mouse is put to death, in 70% alcohol immersion moments later, open the abdominal cavity, get spleen.Push spleen with the syringe nook closing member gently at 200 order Stainless Steel Cloths, make unicellular in woven wire flows into plate (15mL has been housed has contained 10%FBS RPMI-1640).Then with the flushing of the substratum in plate woven wire.Single cell suspension after 400 order woven wires filter, with filtrate in the centrifugal 10min of 200g.Remove supernatant, in precipitation, add 5mL Tris-NH ₄Cl (pH7.2) hangs cell, and room temperature is placed 10min, to remove red corpuscle.The centrifugal 10min of 200g removes supernatant, uses Tris-NH ₄Cl (pH7.2) removes red corpuscle once again.In precipitation, add 5mL at last and contain 10%FBS RPMI-1640, hanged cell, counting cells concentration, it is standby to put ice bath.

(2) mensuration of stimulation index

Splenocyte suspension is diluted to 2 * 10 ⁶Cell/ml adds to 96 orifice plates (0.1mL/ hole), and adds stimulator antigen JEV totivirus albumen or canavaline (Con A) concentration is the substratum 100 μ L of 5 μ g/mL, establishes antigenic stimulation group and non-stimulating group (blank group) simultaneously.Splenocyte is cultivated 4d for 37 ℃ after antigen JEV totivirus albumen or canavaline (Con A) stimulation, measure the increment situation of cell with the MTS method.The MTS method: every porocyte adds 20 μ L MTS, and behind the 5hr, light absorption value or the every hole of measuring each hole in the 492nm place add 30 μ l 10%SDS (containing 0.01M HCl) again, after 37 ℃ of night incubation in the light absorption value in each hole of mensuration, 492nm place.The average OD value of stimulation index (SI)=experimental group/average OD value of blank group.

Get three mouse boosting cells after the immunity and grind the back and carry out T cytositimulation exponential with the MTS method and measure, the SI value of pCMW-2M group be about 2.8 and PBS organizes and carrier pCMW-118L organizes SI value differ significantly (P＜0.05).

7 anti-JEV immune serums are attacked the provide protection of poison to JEV:

PCMW-2M immune group and pCMW-G2-R are organized every group of each mice serum equal-volume mix, again with viral liquid (about 1000LD50) balanced mix after, hatch for 37 ℃ and inoculate suckling mouse in the 1hr hindbrain.Simultaneously with viral liquid and normal mouse serum balanced mix, in contrast.Every group of 10-13 suckling mouse, every inoculation 30 μ L, observe suckling mouse incidence and pathogeneticing characteristic, time, whether dead every day from the inoculation, observed for 3 weeks continuously, and not dead mouse draws neck to put to death.The results are shown in Table 2, simple JEV and JEV+ normal mouse serologic group are all fallen ill, and show as to break away from colony, tic, paralysis, and be all dead in 7 days; And continuous observation of JEV+pCMW-2M immune serum group has 3 death 3 weeks, and all the other are all survived, and are in good condition; These results show that virus attack has the better protecting effect to the pCMW-2M immune serum to JEV.

The anti-JEV immune serum of table 2 is attacked the provide protection experimental result of poison to JEV

Annotate: a is the mouse number of survival; B is the experimental mice sum.

The foundation of the packing cell of embodiment 4, expression JEV structural protein

1, the structure of recombinant eukaryon expression vector pCJECME

With the full-length infectious clone of JEV pBR-JTF is template, goes out the C-prM-E gene 2396bp fragment of JEV by pcr amplification with primer pJEVC1 (SEQID NO:16) and primer dXhoE (SEQ ID NO:17).Pcr amplification product C-prM-E gene through the Bstz17I/XhoI double digestion, is cloned among the carrier pCDNA3.1.Structure comprises the carrier for expression of eukaryon pCJECME of the C-prM-E gene of JEV.

2, the clone of JEV structural protein is expressed in screening

(1) ELISA carries out primary dcreening operation

Extract plasmid pCJECME, transfection BHK-21 cell screens through G418200ug/ml after the Ssp I linearizing.Form cell clone about about 3 weeks, the mono-clonal boundary is clearly demarcated, and compactness is good, and it is chosen to 96 orifice plates, and it is vigorous to grow, and the multinuclear phenomenon does not appear in cell.With monoclonal cell amplification culture to 24 orifice plate of picking, the method for using ELISA detects the expression of structural protein in the cells and supernatant, with the negative contrast of normal BHK-21 cell.Altogether picking 21 strain monoclonal cells, further detect through ELISA, screening has obtained the high proteic positive cell clone of JEV C-prM-E of 10 strain relative expression quantities.

(2) Western Blot identifies

Further detect cell pyrolysis liquid and have or not the proteic expression of E about 53kD by Western Blot, sample preparation: with cell clone CME-4 number and the normal BHK-21 cell protein free OPTI-MEM culture medium culturing of serum-free 48hr, the collecting cell culture supernatant, with micro-evaporating pipe with centrifugal about 1 hour of 5ml supernatant 8000g, be concentrated into 250 μ L, and collect supernatant 50 μ L, add 50 μ L, 2 * loading buffer, add 10 μ L beta-mercaptoethanols, mix and boiled 4 minutes.

To 20 μ L protein sample electrophoresis, voltage 180V when bromine Fen indigo plant flushes with the lower edge of glue, stops electrophoresis glue is gone on the electroporation, changes film 1 hour with 15V voltage, and the sample electricity is gone to nitrocellulose filter with 12%SDS-PAGE.With BSA confining liquid (1%BSA, 0.1%Tween is dissolved in PBS) sealing 1 hour, the anti-JEV of cavy how washed 10 minutes with PBS after 1 hour by anti-reaction, and HRP-goat-anti cavy two resists 1 hour, and 10mL TBS liquid (DAB 6 μ g, H are put into film in PBS flushing 10 minutes at last ₂O ₂10 μ L) develop the color.

The result detects the proteic expression of E that has in the cell clone CME-4 lysate about 53kD by Western Blot, conform to the expection size, no specific band manifests in the normal BHK-21 cell and contrast, and shows the expression that the JEV structural protein are arranged in the clone, the results are shown in Figure 6.Extract cell clone CME-4 genome, with pJEVc1 (SEQ ID NO:16)/dXhoE (SEQ ID NO:17) is primer, genome product with extraction is a template, the total length 2396bp fragment of amplification JEV C-prM-E gene, result such as Fig. 7, can amplify corresponding band in the cell clone genome extract, illustrate that JEV C-prM-E gene successfully is incorporated in the BHK-21 cellular genome.Further finding in the experiment that the expression amount that detects target protein in the cells and supernatant is less than the protein expression in the cell pyrolysis liquid, prompting structure albumen major part concentrates in the cell, and the amount that discharges is less.

3, the stability study of clone

In the going down to posterity for a long time of cell, the phenomenon that goal gene in the host cell gene group partly or entirely lacks can appear being integrated into, show as increase along with passage number, the positive cell rate of expressing target protein reduces gradually, even can occur in the phenomenon that the back positive cell that repeatedly goes down to posterity is all lost.

Though we are through optimizing, preliminary screening has arrived the clone of expressing the JEV structural protein, its stability with can be only as the of paramount importance factor of package cell line by the continuous expression structural protein.So need after cultivating certain hour, further identify the ratio of positive cell, cultured continuously 5 months, go down to posterity more than 100 times after, we can the proteic positive cell percentage situation of expression structure with the how anti-evaluation of the anti-JEV in cavy source through indirect immunofluorescence.As can be seen from Figure 8, the ratio of the proteic cell of expression structure still accounts for more than 50% in the clone that screening obtains, and the result is consistent with initial test.Illustrate that the clone that obtains can more stable and continuous expression JEV structural protein.

Through a series of experimental study, we have finally obtained JEV package cell line preferably, for the acquisition of follow-up pseudovirion is had laid a good foundation.

The acquisition of embodiment 5, pseudovirion and amplification culture

1, the expression of JEV replicon pCMW-2M-EGFP in structural protein clone

After will comprising the JEV replicon carrier pCMW-2M-EGFP linearizing of reporter gene EGFP, transfection JEV structural protein clone CME-4, the expression amount of reporter gene has had significant raising than directly changeing the BHK-21 cell, as can be seen from Figure 9, the different time points of replicon carrier pCMW-2M-EGFP behind transfection BHK-21 cell, the expression amount of EGFP increases along with the prolongation of time, reaches as high as about 20%.But behind transfection package cell line CME-4, the expression amount of EGFP improves, and the cell of expressing EGFP in transfection after 24 hours accounts for about 20%, and positive cell increases to about 30% after 72 hours, and fluorescent signal is stable, and endochylema is expressed.Transfection still had the positive cell rate of 30-40% in 120 hours later on, observed the CME-4 cellular form, and normal cell still is in the great majority, and few cell has the circle of change obscission.

2, the expression of JEV replicon pCMW-2M-LAC in structural protein clone

Behind replicon carrier pCMW-2M-LAC difference transfection package cell line CME-4, CME-17, the expression amount that can observe LacZ is significantly improved, as shown in figure 10, in 12 orifice plates after linearization of the pCMW-2M-LAC plasmid DNA transfection JEV structural protein clone of 1.6 μ g, cell expressing LacZ about 20% is arranged, and cell indigo plant is dyed obviously, and the prompting expression amount is higher.

3, the amplification culture of JEV replicon virion pCMW-2M-LAC in structural protein clone

After replicon carrier plasmid pCMW-2M-LAC linearizing, the clone CME-4 of transfection expression JEV structural protein collected culture supernatant after 4 days, and cells infected is CME-4 and normal BHK-21 cell respectively.Again after 4 days with the former culture supernatant once more cells infected be CME-4 and normal BHK-21 cell.With fixedly original position dyeing of archeocyte, detect the expression of LacZ when infecting.

Replicon carrier pCMW-2M-LAC transfection package cell line CME-4, after 4 days culture supernatant being continued cells infected is, the expression that detects LacZ simultaneously is shown in Figure 11 A, cell expressing LacZ about 50% is arranged in the s-generation clone as can be seen approximately, and see that from the dyeing situation expression amount is suitable with the first-generation.Gathering in the crops this expression supernatant continuation cells infected is CME-4, and the expression of LacZ is shown in Figure 11 B.Positive cell number is more than the s-generation as can be seen, and the equal indigo plant of most of cell is dyed, and relative expression amount is weaker than s-generation cell.After continuing to infect for the third time, the ratio of positive cell descends more obvious, shows that as Figure 11 C expression amount also descends.After first-generation culture supernatant infected normal BHK-21 cells, positive rate did not further improve, and expression amount is lower, shown in Figure 11 D.According to first-generation result of infection, the titre of calculating pseudovirion is 1.2 * 10 ³U/ml, the pseudovirion titre that s-generation result of infection calculates is about 1 * 10 ⁴U/ml.Because the about 3.1kb of LacZ gene, it is the pseudovirion of recombinating that our package cell line can be packaged into it, and when this also pointed out us to utilize the replicon carrier system in the future, length range that can expressed exogenous gene was not less than 3kb at least.

The acquisition of embodiment 6, pseudovirion and amplification culture

1, the preparation of reorganization pseudovirion

Carried out replicon carrier pCMW-2M, pCMW-2M-EGFP, pCMW-2M-LAC transfection package cell line to obtain pseudovirion, the concrete operations flow process is with above-mentioned consistent.

2, the transmission electron microscope observing of JEV pseudovirion

The negative staining electron microscope look of pseudovirion is observed as shown in figure 12, and in the cell conditioned medium of several different pseudovirions, negative staining all can be observed virion, and is how rounded, and diameter between 40-60nm, is consistent with the structure of JEV greatly.And the hollow shape that exists minority to be dispersed in do not have nuclear particle, about the about 20nm of diameter, analyzes the virus-like particle (VLPs) that constitutes into prM-E, owing to do not contain the nucleocapsid structure, so be cavity shape globosity, big or smallly conforms to bibliographical information.

3, RT-PCR identifies pseudovirion

Behind the pseudovirion infection BHK-21 cell with results, with metainfective cell harvesting, extract RNA, obtain cDNA first chain by reverse transcription, with primer CX12 (SEQID NO:18)/P3R (SEQ ID NO:19), CX16a (SEQ ID the NO:20)/genome 7352-8171 sequence of CX16b (SEQ ID NO:21) amplification pseudovirion and the specific fragment of 9316-9479nt sequence, length is respectively 810bp, 164bp.As can be seen from Figure 13, amplify and expect big or small corresponding to band.And BHK-21 cell negative control group fails to amplify the expection band, may contaminated possibility thereby got rid of nutrient solution.The above results has all confirmed to have in the culturing cell generation of pseudovirus.

The experimentation on animals of embodiment 7, JEV pseudovirion

1, the specific antibody level of JEV replicon pseudovirion immune mouse

With the JEV pseudovirion with 5 * 10 ⁴U/ immune mouse, grouping situation see Table 3, two all backs booster immunizations once, and 2 exempt from back two all mouse tails gets blood, and the ELISA method detects the level of E protein specific antibody in the serum.Set up CME-4 cell conditioned medium and PBS contrast simultaneously.After the result shows JEV pseudovirion immune mouse, can produce proteic specific antibody reaction at E, antibody titers reaches 1: 2560 after immune 2 times, and in the CME-4 cell conditioned medium because of containing the E albumen of expression, so also can produce specific at the proteic antibody response of E, the immunity 2 times after antibody titers reach 1: 640.See Figure 14, antibody horizontal and PBS control group significant difference (P＜0.05) in JEV pseudovirus immune group and the package cell line culture supernatant immune group serum.

Table 3 pseudovirion immune mouse grouping situation

2, the mensuration of serum neutralizing antibody level behind the JEV pseudovirion immune mouse

Serum behind JEV pseudovirion and the package cell line culture supernatant immune mouse is carried out the plaque experiment with JEV virus strain effect postoperative infection BHK-21 cell respectively, and 5-7 can observe the generation cytopathy after day under mirror, show as cellular swelling, accumulation.Profile is obviously different with normal cell, and as seen naked eyes have hickie to form, and is to observe to have or not the direct indicator that produces virus.Judge the neutralizing antibody level of serum by the highest serum extent of dilution that reduces by 50% plaque, the result shows that the NAT of the immune serum of JE pseudovirus reaches 1: 128.

3, immune serum is attacked the protection experiment of suckling mouse to JEV

With JEV virus liquid respectively with JEV pseudovirion and package cell line culture supernatant immune mouse after serum effect after 1 hour, intracerebral injection suckling mouse brain tissue.Observed for 3 weeks, the results are shown in Table 4, behind the simple virus injection suckling mouse, all morbidities show as disengaging colony, and are dispirited, all dead in 7 days.And after being added with JEV pseudovirion and package cell line culture supernatant immune serum, virus liquid obviously reduces the attacking ability of suckling mouse, there is mouse disease symptom to occur about 2 weeks, JEV pseudovirion group still has 8 mouse survivals during 3 weeks, package cell line culture supernatant group has 5 mouse survivals, and all in good condition.Demonstrated of the better provide protection of JEV PIPs immune serum to virus attack.

Table 4JEV pseudovirus immune serum is to suckling mouse endogenous protective experimental result

Sequence table

＜110〉Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A

＜120〉a kind of Japanese encephalitis virus JEV replicon vector and application thereof

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gatcgccctt?cccaacagtt?gcgcagcctg?aatggcgaat?ggcgctttgc?ctggtttccg 180

gtaccagaag?cggtgccgga?aagctggctg?gagtgcgatc?ttcctgaggc?cgatactgtc 240

gtcgtcccct?caaactggca?gatgcacggt?tacgatgcgc?ccatctacac?caacgtaacc 300

tatcccatta?cggtcaatcc?gccgtttgtt?cccacggaga?atccgacggg?ttgttactcg 360

ctcacattta?atgttgatga?aagctggcta?caggaaggcc?agacgcgaat?tatttttgat 420

ggcgttaact?cggcgtttca?tctgtggtgc?aacgggcgct?gggtcggtta?cggccaggac 480

agtcgtttgc?cgtctgaatt?tgacctgagc?gcatttttac?gcgccggaga?aaaccgcctc 540

gcggtgatgg?tgctgcgttg?gagtgacggc?agttatctgg?aagatcagga?tatgtggcgg 600

atgagcggca?ttttccgtga?cgtctcgttg?ctgcataaac?cgactacaca?aatcagcgat 660

ttccatgttg?ccactcgctt?taatgatgat?ttcagccgcg?ctgtactgga?ggctgaagtt 720

cagatgtgcg?gcgagttgcg?tgactaccta?cgggtaacag?tttctttatg?gcagggtgaa 780

acgcaggtcg?ccagcggcac?cgcgcctttc?ggcggtgaaa?ttatcgatga?gcgtggtggt 840

tatgccgatc?gcgtcacact?acgtctgaac?gtcgaaaacc?cgaaactgtg?gagcgccgaa 900

atcccgaatc?tctatcgtgc?ggtggttgaa?ctgcacaccg?ccgacggcac?gctgattgaa 960

gcagaagcct?gcgatgtcgg?tttccgcgag?gtgcggattg?aaaatggtct?gctgctgctg 1020

aacggcaagc?cgttgctgat?tcgaggcgtt?aaccgtcacg?agcatcatcc?tctgcatggt 1080

caggtcatgg?atgagcagac?gatggtgcag?gatatcctgc?tgatgaagca?gaacaacttt 1140

aacgccgtgc?gctgttcgca?ttatccgaac?catccgctgt?ggtacacgct?gtgcgaccgc 1200

tacggcctgt?atgtggtgga?tgaagccaat?attgaaaccc?acggcatggt?gccaatgaat 1260

cgtctgaccg?atgatccgcg?ctggctaccg?gcgatgagcg?aacgcgtaac?gcgaatggtg 1320

cagcgcgatc?gtaatcaccc?gagtgtgatc?atctggtcgc?tggggaatga?atcaggccac 1380

ggcgctaatc?acgacgcgct?gtatcgctgg?atcaaatctg?tcgatccttc?ccgcccggtg 1440

cagtatgaag?gcggcggagc?cgacaccacg?gccaccgata?ttatttgccc?gatgtacgcg 1500

cgcgtggatg?aagaccagcc?cttcccggct?gtgccgaaat?ggtccatcaa?aaaatggctt 1560

tcgctacctg?gagagacgcg?cccgctgatc?ctttgcgaat?acgcccacgc?gatgggtaac 1620

agtcttggcg?gtttcgctaa?atactggcag?gcgtttcgtc?agtatccccg?tttacagggc 1680

ggcttcgtct?gggactgggt?ggatcagtcg?ctgattaaat?atgatgaaaa?cggcaacccg 1740

tggtcggctt?acggcggtga?ttttggcgat?acgccgaacg?atcgccagtt?ctgtatgaac 1800

ggtctggtct?ttgccgaccg?cacgccgcat?ccagcgctga?cggaagcaaa?acaccagcag 1860

cagtttttcc?agttccgttt?atccgggcaa?accatcgaag?tgaccagcga?atacctgttc 1920

cgtcatagcg?ataacgagct?cctgcactgg?atggtggcgc?tggatggtaa?gccgctggca 1980

agcggtgaag?tgcctctgga?tgtcgctcca?caaggtaaac?agttgattga?actgcctgaa 2040

ctaccgcagc?cggagagcgc?cgggcaactc?tggctcacag?tacgcgtagt?gcaaccgaac 2100

gcgaccgcat?ggtcagaagc?cgggcacatc?agcgcctggc?agcagtggcg?tctggcggaa 2160

aacctcagtg?tgacgctccc?cgccgcgtcc?cacgccatcc?cgcatctgac?caccagcgaa 2220

atggattttt?gcatcgagct?gggtaataag?cgttggcaat?ttaaccgcca?gtcaggcttt 2280

ctttcacaga?tgtggattgg?cgataaaaaa?caactgctga?cgccgctgcg?cgatcagttc 2340

acccgtgcac?cgctggataa?cgacattggc?gtaagtgaag?cgacccgcat?tgaccctaac 2400

gcctgggtcg?aacgctggaa?ggcggcgggc?cattaccagg?ccgaagcagc?gttgttgcag 2460

tgcacggcag?atacacttgc?tgatgcggtg?ctgattacga?ccgctcacgc?gtggcagcat 2520

caggggaaaa?ccttatttat?cagccggaaa?acctaccgga?ttgatggtag?tggtcaaatg 2580

gcgattaccg?ttgatgttga?agtggcgagc?gatacaccgc?atccggcgcg?gattggcctg 2640

aactgccagc?tggcgcaggt?agcagagcgg?gtaaactggc?tcggattagg?gccgcaagaa 2700

aactatcccg?accgccttac?tgccgcctgt?tttgaccgct?gggatctgcc?attgtcagac 2760

atgtataccc?cgtacgtctt?cccgagcgaa?aacggtctgc?gctgcgggac?gcgcgaattg 2820

aattatggcc?cacaccagtg?gcgcggcgac?ttccagttca?acatcagccg?ctacagtcaa 2880

cagcaactga?tggaaaccag?ccatcgccat?ctgctgcacg?cggaagaagg?cacatggctg 2940

aatatcgacg?gtttccatat?ggggattggt?ggcgacgact?cctggagccc?gtcagtatcg 3000

gcggaattcc?agctgagcgc?cggtcgctac?cattaccagt?tggtctggtg?tcaaaaa 3057

<210>16

<211>35

<212>DNA

＜213〉artificial sequence

<400>16

accgtatacg?ccgccaccat?gactaaaaaa?ccagg 35

<210>17

<211>38

<212>DNA

＜213〉artificial sequence

<400>17

tagctcgagc?tattaagcat?gcacattggt?cgctaaga 38

<210>18

<211>18

<212>DNA

＜213〉artificial sequence

<400>18

gaatgccgtt?gttgacgg 18

<210>19

<211>31

<212>DNA

＜213〉artificial sequence

<400>19

taggacgcgt?agtgtgcgtt?gttcttctac?t 31

<210>20

<211>18

<212>DNA

＜213〉artificial sequence

<400>20

gagggaaaac?catctgcg 18

<210>21

<211>21

<212>DNA

＜213〉artificial sequence

<400>21

ttgatcttct?cttgatatca?c 21

<210>22

<211>14122

<212>DNA

＜213〉artificial sequence

<400>22

gacagtaaga?cgggtaagcc?tgttgatgat?accgctgcct?tactgggtgc?attagccagt 60

ctgaatgacc?tgtcacggga?taatccgaag?tggtcagact?ggaaaatcag?agggcaggaa 120

ctgcagaaca?gcaaaaagtc?agatagcacc?acatagcaga?cccgccataa?aacgccctga 180

gaagcccgtg?acgggctttt?cttgtattat?gggtagtttc?cttgcatgaa?tccataaaag 240

gcgcctgtag?tgccatttac?ccccattcac?tgccagagcc?gtgagcgcag?cgaactgaat 300

gtcacgaaaa?agacagcgac?tcaggtgcct?gatggtcgga?gacaaaagga?atattcagcg 360

atttgcccga?gcttgcgagg?gtgctactta?agcctttagg?gttttaaggt?ctgttttgta 420

gaggagcaaa?cagcgtttgc?gacatccttt?tgtaatactg?cggaactgac?taaagtagtg 480

agttatacac?agggctggga?tctattcttt?ttatcttttt?ttattctttc?tttattctat 540

aaattataac?cacttgaata?taaacaaaaa?aaacacacaa?aggtctagcg?gaatttacag 600

agggtctagc?agaatttaca?agttttccag?caaaggtcta?gcagaattta?cagataccca 660

caactcaaag?gaaaaggact?agtaattatc?attgactagc?ccatctcaat?tggtatagtg 720

attaaaatca?cctagaccaa?ttgagatgta?tgtctgaatt?agttgttttc?aaagcaaatg 780

aactagcgat?tagtcgctat?gacttaacgg?agcatgaaac?caagctaatt?ttatgctgtg 840

tggcactact?caaccccacg?attgaaaacc?ctacaaggaa?agaacggacg?gtatcgttca 900

cttataacca?atacgctcag?atgatgaaca?tcagtaggga?aaatgcttat?ggtgtattag 960

ctaaagcaac?cagagagctg?atgacgagaa?ctgtggaaat?caggaatcct?ttggttaaag 1020

gctttgagat?tttccagtgg?acaaactatg?ccaagttctc?aagcgaaaaa?ttagaattag 1080

tttttagtga?agagatattg?ccttatcttt?tccagttaaa?aaaattcata?aaatataatc 1140

tggaacatgt?taagtctttt?gaaaacaaat?actctatgag?gatttatgag?tggttattaa 1200

aagaactaac?acaaaagaaa?actcacaagg?caaatataga?gattagcctt?gatgaattta 1260

agttcatgtt?aatgcttgaa?aataactacc?atgagtttaa?aaggcttaac?caatgggttt 1320

tgaaaccaat?aagtaaagat?ttaaacactt?acagcaatat?gaaattggtg?gttgataagc 1380

gaggccgccc?gactgatacg?ttgattttcc?aagttgaact?agatagacaa?atggatctcg 1440

taaccgaact?tgagaacaac?cagataaaaa?tgaatggtga?caaaatacca?acaaccatta 1500

catcagattc?ctacctacat?aacggactaa?gaaaaacact?acacgatgct?ttaactgcaa 1560

aaattcagct?caccagtttt?gaggcaaaat?ttttgagtga?catgcaaagt?aagcatgatc 1620

tcaatggttc?gttctcatgg?ctcacgcaaa?aacaacgaac?cacactagag?aacatactgg 1680

ctaaatacgg?aaggatctga?ggttcttatg?gctcttgtat?ctatcagtga?agcatcaaga 1740

ctaacaaaca?aaagtagaac?aactgttcac?cgttacatat?caaagggaaa?actgtccata 1800

tgcacagatg?aaaacggtgt?aaaaaagata?gatacatcag?agcttttacg?agtttttggt 1860

gcattcaaag?ctgttcacca?tgaacagatc?gacaatgtaa?cagatgaaca?gcatgtaaca 1920

cctaatagaa?caggtgaaac?cagtaaaaca?aagcaactag?aacatgaaat?tgaacacctg 1980

agacaacttg?ttacagctca?acagtcacac?atagacagcc?tgaaacaggc?gatgctgctt 2040

atcgaatcaa?agctgccgac?aacacgggag?ccagtgacgc?ctcccgtggg?gaaaaaatca 2100

tggcaattct?ggaagaaata?gcgccattcg?ccattcaggc?tgcgcaactg?ttgggaaggg 2160

cgatcggtgc?gggcctcttc?gctattacgc?cagctggcga?aagggggatg?tgctgcaagg 2220

cgattaagtt?gggtaacgcc?agggttttcc?cagtcacgac?gttgtaaaac?gacggccagt 2280

gccaagcttg?catgcgttga?cattgattat?tgactagtta?ttaatagtaa?tcaattacgg 2340

ggtcattagt?tcatagccca?tatatggagt?tccgcgttac?ataacttacg?gtaaatggcc 2400

cgcctggctg?accgcccaac?gacccccgcc?cattgacgtc?aataatgacg?tatgttccca 2460

tagtaacgcc?aatagggact?ttccattgac?gtcaatgggt?ggagtattta?cggtaaactg 2520

cccacttggc?agtacatcaa?gtgtatcata?tgccaagtac?gccccctatt?gacgtcaatg 2580

acggtaaatg?gcccgcctgg?cattatgccc?agtacatgac?cttatgggac?tttcctactt 2640

ggcagtacat?ctacgtatta?gtcatcgcta?ttaccatggt?gatgcggttt?tggcagtaca 2700

tcaatgggcg?tggatagcgg?tttgactcac?ggggatttcc?aagtctccac?cccattgacg 2760

tcaatgggag?tttgttttgg?caccaaaatc?aacgggactt?tccaaaatgt?cgtaacaact 2820

ccgccccatt?gacgcaaatg?ggcggtaggc?gtgtacggtg?ggaggtctat?ataagcagag 2880

ctctgtcgac?taatacgact?cactatagag?aagtttatct?gtgtgaactt?cttggcttag 2940

tatcgtagag?aagaatcgag?agattagtgc?agtttaaaca?gttttttaga?acggaagata 3000

accatgacta?aaaaaccagg?agggcccggt?aaaaaccggg?ctatcaatat?gctgaaacgc 3060

ggcctacccc?gcgtattccc?actagtggga?gtgaagaggg?tagtaatgag?cttgttggac 3120

ggcagagggc?cagtacgttt?cgtgctggct?cttatcacgt?tcttcaagtt?tacagcatta 3180

gccccgacca?aggcgctttc?aggccgatgg?aaagcagtgg?aaaagagtgt?ggcaatgaaa 3240

catcttacta?gtttcaaacg?agaacttgga?acactcattg?acgccgtgaa?caagcggggc 3300

agaaagcaaa?acaaaagagg?aggaaatgaa?ggctcaatca?tgtggctcgc?gagcttggca 3360

gttgtcatag?cttgtgcagg?agccctcgag?acgtgtacac?agactttgaa?ttttgacctt 3420

ctcaagttgg?cgggagacgt?cgagtccaac?cctgggccga?ttgctttggc?cttcttagcc 3480

acaggaggtg?tgctcgtgtt?cttagcgacc?aatgtgcatg?ctgacactgg?atgtgccatt 3540

gacatcacaa?gaaaagagat?gagatgtgga?agtggcatct?tcgtgcacaa?cgacgtggaa 3600

gcctgggtgg?ataggtataa?atatttgcca?gaaacgccca?gatccctagc?gaagatcgtc 3660

cacaaagcgc?acaaggaagg?cgtgtgcgga?gtcagatctg?tcactagact?ggagcaccaa 3720

atgtgggaag?ccgtaaggga?cgaattgaac?gtcctgctca?aagagaatgc?agtggacctc 3780

agtgtggttg?tgaacaagcc?cgtgggaaga?tatcgctcag?cccctaaacg?cctatccatg 3840

acgcaagaga?agtttgaaat?gggctggaaa?gcatggggaa?aaagcatcct?ctttgccccg 3900

gaattggcta?actccacatt?tgtcgtagat?ggacctgaga?caaaggaatg?ccctgatgag 3960

cacagagctt?ggaacagcat?gcaaatcgaa?gacttcggct?ttggcatcac?atcaacccgt 4020

gtgtggctga?aaattagaga?ggagagcact?gacgagtgtg?atggagcgat?cataggcacg 4080

gctgtcaaag?gacatgtggc?agtccatagt?gacttgtcgt?actggattga?gagtcgctac 4140

aacgacacat?ggaaacttga?gagggcagtc?tttggagagg?tcaaatcttg?cacttggcca 4200

gagacacaca?ccctttgggg?agatgatgtt?gaggaaagtg?aactcatcat?tccgcacacc 4260

atagccggac?caaaaagcaa?gcacaatcgg?agggaagggt?ataagacaca?aaaccaggga 4320

ccttgggatg?agaatggcat?agtcttggac?tttgattatt?gcccagggac?aaaagtcacc 4380

attacagagg?attgtagcaa?gagaggccct?tcggtcagaa?ccactactga?cagtggaaag 4440

ttgatcactg?actggtgctg?tcgcagttgc?tcccttccgc?ccctacgatt?ccggacagaa 4500

aatggctgct?ggtacggaat?ggaaatcaga?cctgttatgc?atgatgaaac?aacactcgtc 4560

agatcacagg?ttcatgcttt?caaaggtgaa?atggttgacc?cttttcagct?gggccttctg 4620

gtgatgtttc?tggccaccca?ggaagtcctt?cgcaagaggt?ggacggccag?attgaccatt 4680

cctgcggttt?tgggggtcct?acttgtgctg?atgcttgggg?gtatcactta?cactgatttg 4740

gcgaggtatg?tggtgctagt?cgctgctgct?ttcgcagagg?ccaacagtgg?aggagacgtc 4800

ctgcaccttg?ctttgattgc?tgtttttaag?atccaaccag?catttttagt?gatgaacatg 4860

cttagcacga?gatggacgaa?ccaagaaaac?gtggttctgg?tcctaggggc?tgcctttttc 4920

caattggcct?cagtagatct?gcaaatagga?gtccacggaa?tcctgaatgc?cgccgctata 4980

gcatggatga?ttgtccgagc?gatcaccttc?cccacaacct?cctccgtcac?catgccagtc 5040

ttagcgcttc?taactccggg?gatgagggct?ctatacctag?acacttacag?aatcatcctc 5100

ctcgtcatag?ggatttgctc?cctgctgcac?gagaggaaaa?agaccatggc?gaaaaagaaa 5160

ggagctgtac?tcttgggctt?agcgctcaca?tccactggat?ggttctcgcc?caccactata 5220

gctgccggac?taatggtctg?caacccaaac?aagaagagag?ggtggccagc?tactgagttt 5280

ttgtcggcag?ttggattgat?gtttgccatc?gtaggtggtt?tggccgagtt?ggatattgaa 5340

tccatgtcaa?tacccttcat?gctggcaggt?ctcatggcag?tgtcctacgt?ggtgtcagga 5400

aaagcaacag?atatgtggct?tgaacgggcc?gccgacatca?gctgggatat?gggtgctgca 5460

atcacaggaa?gcagtcggag?gctggatgtg?aaactggatg?atgacggaga?ttttcacttg 5520

attgatgatc?ccggtgttcc?atggaaggtc?tgggtcctgc?gcatgtcttg?cattggctta 5580

gccgccctca?cgccttgggc?catcgttccc?gccgctttcg?gttattggct?cactttaaaa 5640

acaacaaaaa?gagggggcgt?gttttgggac?acgccatccc?caaaacctat?ggcacacaac 5700

tagagttgct?caaaaggaga?caccactaca?ggagtctacc?gaattatggc?tagagggatt 5760

cttggcactt?accaggccgg?cgtcggagtc?atgtacgaga?atgttttcca?cacactatgg 5820

cacacaacta?gaggagcagc?cattgtgagt?ggagaaggaa?aattgacgcc?atactggggt 5880

agtgtgaaag?aagaccgcat?agcttacgga?ggcccatgga?ggtttgaccg?aaaatggaat 5940

ggaacagatg?acgtgcaagt?gatcgtggta?gaaccgggga?agggcgcagt?aaacatccag 6000

acaaaaccag?gagtgtttcg?gactcccttc?ggggaggttg?gggctgttag?tctggattac 6060

ccgcgaggaa?catccggctc?acccattctg?gattccaatg?gagacattat?aggcctatac 6120

ggcaatggag?ttgagcttgg?cgatggctca?tacgtcagcg?ccatcgtgca?gggtgaccgt 6180

caggaggaac?cagtcccaga?agcttacacc?ccaaacatgt?tgagaaagag?acagatgact 6240

gtgctagatt?tgcaccctgg?ttcagggaaa?accaggaaaa?ttctgccaca?aataattaag 6300

gacgctatcc?agcagcgcct?aagaacagct?gtgttggcac?cgacgcgggt?ggtagcagca 6360

gaaatggcag?aagttttgag?agggctccca?gtacgatatc?aaacttcagc?agtgcagaga 6420

gagcaccaag?ggaatgaaat?agtggatgtg?atgtgccacg?ccactctgac?ccatagactg 6480

atgtcaccga?acagagtgcc?caactacaac?ctatttgtca?tggatgaagc?tcatttcacc 6540

gacccagcca?gtatagccgc?acgaggatac?attgctacca?aggtggaatt?aggggaggca 6600

gcagccatct?ttatgacagc?gaccccgcct?ggaaccacgg?atccttttcc?tgactcaaat 6660

gccccaatcc?atgatttgca?agatgagata?ccagacaggg?catggagcag?tggatacgaa 6720

tggatcacag?aatatgcggg?taaaaccgtg?tggtttgtgg?cgagcgtaaa?aatggggaat 6780

gagattgcaa?tgtgcctcca?aagagcgggg?aaaaaggtca?tccaactcaa?ccgcaagtcc 6840

tatgacacag?aatacccaaa?atgtaagaat?ggagactggg?attttgtcat?taccaccgac 6900

atctctgaaa?tgggggccaa?cttcggtgcg?agcagggtca?tcgactgtag?aaagagcgtg 6960

aaacccacca?tcttagaaga?gggagaaggc?agagtcatcc?tcggaaaccc?atctcccata 7020

accagtgcaa?gcgcagctca?acggaggggc?agagtaggca?gaaaccccaa?tcaagttgga 7080

gatgaatacc?actatggggg?ggctaccagt?gaagatgaca?gtaacctagc?ccattggaca 7140

gaggcaaaga?tcatgttaga?caacatacac?atgcccaatg?gactggtggc?ccagctctat 7200

ggaccagaga?gggaaaaggc?tttcacaatg?gatggcgaat?accgtctcag?aggtgaagaa 7260

aagaaaaact?tcttagagct?gcttaggacg?gctgacctcc?cggtgtggct?ggcctacaag 7320

gtggcgtcca?atggcattca?gtacaccgac?agaaagtggt?gttttgatgg?gccgcgtacg 7380

aatgccatac?tggaggacaa?caccgaggta?gagatagtca?cccggatggg?tgagaggaaa 7440

atcctcaagc?cgagatggct?tgatgcaaga?gtttatgcag?atcaccaggc?cctcaagtgg 7500

ttcaaagact?ttgcagcagg?gaagagatca?gccgttagct?tcatagaggt?gctcggtcgc 7560

atgcctgagc?atttcatggg?aaagacgcgg?gaagctttag?acaccatgta?cttggttgca 7620

acggctgaga?aaggtgggaa?agcacaccga?atggctctcg?aagagctgcc?agatgcactg 7680

gaaaccatca?cacttattgt?cgccattact?gtgatgacag?gaggattctt?cctactaatg 7740

atgcagcgaa?agggtatagg?gaagatgggt?cttggagctc?tagtgctcac?actagctacc 7800

ttcttcctgt?gggcggcaga?ggttcctgga?accaaaatag?cagggaccct?gctgatcgcc 7860

ctgctgctga?tggtggttct?catcccagaa?ccggaaaaac?agaggtcaca?gacagataac 7920

caactggcgg?tgtttctcat?ctgtgtcttg?accgtggttg?gagtggtggc?agcaaacgag 7980

tacgggatgc?tagaaaaaac?caaagcggat?ctcaagagca?tgtttggcgg?aaagacgcag 8040

gcatcaggac?tgactggatt?gccaagcatg?gcactggacc?tgcgtccagc?cacagcctgg 8100

gcactgtatg?gggggagcac?agtcgtgcta?acccctcttc?tgaagcacct?gatcacgtcg 8160

gaatacgtca?ccacatcgct?agcttcaatt?aactcacaag?ctggctcatt?attcgtcttg 8220

ccacgaggcg?tgccttttac?cgacctagac?ttgactgttg?gcctcgtctt?ccttggctgt 8280

tggggtcaag?tcaccctcac?aacgtttctg?acagccatgg?ttctggcgac?acttcactat 8340

gggtacatgc?tccctggatg?gcaagcagaa?gcactcaggg?ctgcccagag?aaggacagcg 8400

gctggaataa?tgaagaatgc?cgttgttgac?ggaatggtcg?ccactgatgt?gcctgaactg 8460

gaaaggacta?ctcctctgat?gcaaaagaaa?gtcggacagg?tgctcctcat?aggggtaagc 8520

gtggcagcgt?tcctcgtcaa?ccctaatgtc?accactgtga?gagaagcagg?ggtgttggtg 8580

acggcggcta?cgcttacttt?gtgggacaat?ggagccagtg?ccgtttggaa?ttccaccaca 8640

gccacgggac?tctgccatgt?catgcgaggt?agctacctgg?ctggaggctc?cattgcttgg 8700

actctcatca?agaacgctga?taagccctcc?ttgaaaaggg?gaaggcctgg?gggcaggacg 8760

ctaggggagc?agtggaagga?aaaactaaat?gccatgagta?gagaagagtt?ttttaaatac 8820

cggagagagg?ccataatcga?ggtggaccgc?actgaagcac?gcagggccag?acgtgaaaat 8880

aacatagtgg?gaggacatcc?ggtttcgcga?ggctcagcaa?aactccgttg?gctcgtggag 8940

aaaggatttg?tctcgccaat?aggaaaagtc?attgatctag?ggtgtgggcg?tggaggatgg 9000

agctactacg?cagcaaccct?gaagaaggtc?caggaagtca?gaggatacac?gaaaggtggg 9060

gcgggacatg?aagaaccgat?gctcatgcag?agctacggct?ggaacctggt?ctccctgaag 9120

agtggagtgg?acgtgtttta?caaaccttca?gagcccagtg?ataccctgtt?ctgtgacata 9180

ggggaatcct?ccccaagtcc?agaagtagaa?gaacaacgca?cactacgcgt?cctagagatg 9240

acatctgact?ggttgcaccg?aggacctaga?gagttctgca?ttaaagttct?ctgcccttac 9300

atgcccaagg?ttatagaaaa?aatggaagtt?ctgcagcgtc?gcttcggagg?tgggctagtg 9360

cgtctccccc?tgtcccgaaa?ctccaatcac?gagatgtatt?gggttagtgg?agccgctggc 9420

aatgtggtgc?acgctgtgaa?catgaccagc?caggtattac?tggggcgaat?ggatcgcaca 9480

gtgtggagag?ggccaaagta?tgaggaagat?gtcaacctag?ggagcggaac?aagagccgtg 9540

ggaaagggag?aagtccatag?caatcaggag?aaaatcaaga?agagaatcca?gaagcttaaa 9600

gaagaattcg?ccacaacgtg?gcacaaagac?cctgagcatc?cataccgcac?ttggacatac 9660

cacggaagct?atgaagtgaa?ggctactggc?tcagccagct?ctctcgtcaa?cggagtggtg 9720

aagctcatga?gcaaaccttg?ggacgccatt?gccaacgtca?ccaccatggc?catgactgac 9780

accacccctt?ttggacagca?aagagttttc?aaggagaaag?ttgacacgaa?ggctcctgag 9840

ccaccagctg?gagccaagga?agtgctcaac?gagaccacca?actggctgtg?ggcctacttg 9900

tcacgggaaa?aaagaccccg?cttgtgcacc?aaggaagaat?tcattaagaa?agttaacagc 9960

aacgcggctc?ttggagcagt?gttcgctgaa?cagaatcaat?ggagcacggc?gcgtgaggct 10020

gtggatgacc?cgcggttttg?ggagatggtt?gatgaagaga?gggaaaacca?tctgcgagga 10080

gagtgtcaca?catgtatcta?caacatgatg?ggaaaaagag?agaagaagcc?tggagagttt 10140

ggaaaagcta?aaggaagcag?ggccatttgg?ttcatgtggc?ttggagcacg?gtaccttgag 10200

tttgaagctt?tggggttcct?gaatgaagac?cattggctga?gccgagagaa?ttcaggaggt 10260

ggagtggaag?gctcaggcgt?ccaaaagctg?ggatacatcc?tccgtgacat?agcaggaaag 10320

caaggaggga?aaatgtacgc?tgatgatacc?gccgggtggg?acactagaat?taccagaact 10380

gatttagaaa?atgaagctaa?ggtactggag?ctcctagacg?gtgaacaccg?catgctcgcc 10440

cgagccataa?ttgaactgac?ttacaggcac?aaagtggtca?aggtcatgag?acctgcagca 10500

gaaggaaaga?ccgtgatgga?cgtgatatca?agagaagatc?aaagggggag?tggacaggtg 10560

gtcacttatg?ctcttaacac?tttcacgaac?atcgctgtcc?agctcgtcag?gctgatggag 10620

gctgaggggg?tcattggacc?acaacacttg?gaacatctac?ctaggaaaaa?caagatagct 10680

gtcaggacct?ggctctttga?gaatggagag?gagagagtga?ccaggatggc?gatcagcgga 10740

gacgactgtg?ccgtcaaacc?gctggacgac?agattcgcca?cagccctcca?cttcctcaac 10800

gcaatgtcaa?aggtcagaaa?agacatccag?gaatggaagc?cttcgcatgg?ctggcacgat 10860

tggcagcaag?ttcccttctg?ttctaaccat?tttcaggaga?ttgtgatgaa?agatggaagg 10920

agtatagttg?tcccgtgcag?aggacaggat?gagctgatag?gcagggctcg?catctctcca 10980

ggagctggat?ggaatgtgaa?ggacacagct?tgcctggcca?aagcatatgc?acagatgtgg 11040

ctactcctat?acttccatcg?cagggacttg?cgtctcatgg?caaatgcgat?ttgctcagca 11100

gtgccagtag?attgggtgcc?cacaggcagg?acatcctggt?caatacactc?gaaaggagag 11160

tggatgacca?cggaagacat?gctgcaggtc?tggaacagag?tttggattga?agaaaatgaa 11220

tggatgatgg?acaagactcc?aatcacaagc?tggacagacg?ttccgtatgt?gggaaagcgc 11280

gaggacatct?ggtgtggcag?cctcatcgga?acgcgatcca?gagcaacctg?ggctgagaac 11340

atctatgcgg?cgataaacca?ggttagagct?gtcattggga?aagaaaatta?tgttgactac 11400

atgacctcac?tcaggagata?cgaagacgtc?ttgatccagg?aagacagggt?catctagtgt 11460

gatttaaggt?agaaaagtag?actatgtaaa?caatgtaaat?gagaaaatgc?atgcatatgg 11520

agtcaggcca?gcaaaagctg?ccaccggata?ctgggtagac?ggtgctgcct?gcgtctcagt 11580

cccaggagga?ctgggttaac?aaatctgaca?acagaaagtg?agaaagccct?cagaaccgtc 11640

tcggaagtag?gtccctgctc?actggaagtt?gaaagaccaa?cgtcaggcca?caaatttgtg 11700

ccactccgct?agggagtgcg?gcctgcgcag?ccccaggagg?actgggttac?caaagccgtt 11760

gaggccccca?cggcccaagc?ctcgtctagg?atgcaataga?cgaggtgtaa?ggactagagg 11820

ttagaggaga?ccccgtggaa?acaacaacat?gcggcccaag?ccccctcgaa?gctgtagagg 11880

aggtggaagg?actagaggtt?agaggagacc?ccgcatttgc?atcaaacagc?atattgacac 11940

ctgggaatag?actgggagat?cttctgctct?atctcaacat?cagctactag?gcacagagcg 12000

ccgaagtatg?tagctggtgg?tgaggaagaa?cacaggatct?gggtcggcat?ggcatctcca 12060

cctcctcgcg?gtccgacctg?ggctacttcg?gtaggctaag?ggagaagaac?ttgtttattg 12120

cagcttataa?tggttacaaa?taaagcaata?gcatcacaaa?tttcacaaat?aaagcatttt 12180

tttcactgca?ttctagttgt?ggtttgtcca?aactcatcaa?tgtatcttat?catgtcttct 12240

agaaagaatt?cgtaatcatg?gtcatagctg?tttcctgtgt?gaaattgtta?tccgctcaca 12300

attccacaca?acatacgagc?cggaagcata?aagtgtaaag?cctggggtgc?ctaatgagtg 12360

agctaactca?cattaattgc?gttgcgctca?ctgcccgctt?tccagtcggg?aaacctgtcg 12420

tgccagctgc?attaatgaat?cggccaacgc?gcggggagag?gcggtttgcg?tattgggcgc 12480

tttctcaatg?ctcacgctgt?aggtatctca?gttcggtgta?ggtcgttcgc?tccaagctgg 12540

gctgtgtgca?cgaacccccc?gttcagcccg?accgctgcgc?cttatccggt?aactatcgtc 12600

ttgagtccaa?cccggtaaga?cacgacttat?cgccactggc?agcagccact?ggtaacagga 12660

ttagcagagc?gaggtatgta?ggcggtgcta?cagagttctt?gaagtggtgg?cctaactacg 12720

gctacactag?aaggacagta?tttggtatct?gcgctctgct?gaagccagtt?accttcggaa 12780

aaagagttgg?tagctcttga?tccggcaaac?aaaccaccgc?tggtagcggt?ggtttttttg 12840

tttgcaagca?gcagattacg?cgcagaaaaa?aaggatctca?agaagatcct?ttgatctttt 12900

ctacggggtc?tgacgctcag?tggaacgaaa?actcacgtta?agggattttg?gtcatgagat 12960

tatcaaaaag?gatcttcacc?tagatccttt?taaattaaaa?atgaagtttt?aaatcaatct 13020

aaagtatata?tgagtaaact?tggtctgaca?gttaccaatg?cttaatcagt?gaggcaccta 13080

tctcagcgat?ctgtctattt?cgttcatcca?tagttgcctg?actccccgtc?gtgtagataa 13140

ctacgatacg?ggagggctta?ccatctggcc?ccagtgctgc?aatgataccg?cgagacccac 13200

gctcaccggc?tccagattta?tcagcaataa?accagccagc?cggaagggcc?gagcgcagaa 13260

gtggtcctgc?aactttatcc?gcctccatcc?agtctattaa?ttgttgccgg?gaagctagag 13320

taagtagttc?gccagttaat?agtttgcgca?acgttgttgc?cattgctgca?ggcatcgtgg 13380

tgtcacgctc?gtcgtttggt?atggcttcat?tcagctccgg?ttcccaacga?tcaaggcgag 13440

ttacatgatc?ccccatgttg?tgcaaaaaag?cggttagctc?cttcggtcct?ccgatcgttg 13500

tcagaagtaa?gttggccgca?gtgttatcac?tcatggttat?ggcagcactg?cataattctc 13560

ttactgtcat?gccatccgta?agatgctttt?ctgtgactgg?tgagtactca?accaagtcat 13620

tctgagaata?gtgtatgcgg?cgaccgagtt?gctcttgccc?ggcgtcaaca?cgggataata 13680

ccgcgccaca?tagcagaact?ttaaaagtgc?tcatcattgg?aaaacgttct?tcggggcgaa 13740

aactctcaag?gatcttaccg?ctgttgagat?ccagttcgat?gtaacccact?cgtgcaccca 13800

actgatcttc?agcatctttt?actttcacca?gcgtttctgg?gtgagcaaaa?acaggaaggc 13860

aaaatgccgc?aaaaaaggga?ataagggcga?cacggaaatg?ttgaatactc?atactcttcc 13920

tttttcaata?ttattgaagc?atttatcagg?gttattgtct?catgagcgga?tacatatttg 13980

aatgtattta?gaaaaataaa?caaatagggg?ttccgcgcac?atttccccga?aaagtgccac 14040

ctgacgtcta?agaaaccatt?attatcatga?cattaaccta?taaaaatagg?cgtatcacga 14100

ggccctttcg?tcttcaagaa?tt 14122

Claims (19)

1. JEV replicon carrier, it is a framework construction with the japanese encephalitis virus genome, whole preM genes and the part E gene of described replicon carrier disappearance JEV.

2. replicon carrier as claimed in claim 1 is characterized in that, described replicon carrier comprises the following element that can be operatively connected: promoter sequence; 5 ' end non-coding region; The C protein-coding region; Multiple clone site; The NS1 signal peptide coding region; The Nonstructural Protein coding region; 3 ' end non-coding region.

Such as claim 2 the replicon carrier of book, it is characterized in that described promotor is CMV promotor or T7 promotor.

4. as each described replicon carrier of claim 1～3, it is characterized in that described japanese encephalitis virus is the SA14-14-2 virus strain.

5. as each described replicon carrier of claim 1～3, it is characterized in that, hold in 3 ' of multiple clone site to have 3 ' end releasing member perhaps before described NS1 protein signal peptide-coding region, have 5 ' end releasing member.

6. replicon carrier as claimed in claim 5 is characterized in that, described releasing member is the nucleotide sequence of coding foot and mouth disease virus self lytic enzyme.

7. as each described replicon carrier of claim 1～3, it is characterized in that the nucleotide sequence of described replicon carrier is shown in SEQ ID NO.22.

8. recombinant vectors, it is that each described JEV replicon carrier of claim 1～6 and foreign gene reorganization obtain.

9. recombinant vectors as claimed in claim 8 is characterized in that, described foreign gene is the gene of coding reporter protein or the gene of coding immunogenic protein.

10. recombinant vectors as claimed in claim 9 is characterized in that, the immunogenic protein of described immunogenic protein for expressing in eukaryotic cell.

11. a carrier for expression of eukaryon of expressing the japanese encephalitis virus structural protein, it can pack JEV pseudovirion with each described JEV replicon carrier of claim 1～7 or each described recombinant vectors cotransfection of claim 8～10.

12. carrier for expression of eukaryon as claimed in claim 11 is characterized in that, described expression vector is pCJECME.

13. contain the clone of claim 11 or 12 described carrier for expression of eukaryon.

14. clone as claimed in claim 13, it is the BHK-21 cells of pCJECME stable transfection.

15. a JEV packaging system, it comprises: 1) each described JEV replicon carrier of claim 1～7 or each described recombinant vectors of claim 8～10; 2) claim 13 or 14 described clones.

16. a JEV pseudovirion, it is to be obtained by the described JEV packaging system packing of claim 15.

17. a method for preparing the described JEV pseudovirion of claim 16, it imports in claim 13 or the 14 described clones each described JEV replicon of claim 1～7 or each described recombinant vectors of claim 8～10 through the packing acquisition.

18. each described JEV replicon of claim 1～7 or each described recombinant vectors of claim 8～10, claim 11 or 12 described carrier for expression of eukaryon, claim 13 or 14 described clones or the described JEV packaging system of claim 15 application in preparation amplicon dna vaccine.

19. vaccine that contains the described JEV pseudovirion of claim 16.

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