CN102600481A - Adenovirus (rAdV)/Japanese encephalitis virus (JEV) replicon embedding carrier hog cholera vaccine and application of rAdV/ JEV replicon embedding carrier hog cholera vaccine - Google Patents
Adenovirus (rAdV)/Japanese encephalitis virus (JEV) replicon embedding carrier hog cholera vaccine and application of rAdV/ JEV replicon embedding carrier hog cholera vaccine Download PDFInfo
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- CN102600481A CN102600481A CN2012100735297A CN201210073529A CN102600481A CN 102600481 A CN102600481 A CN 102600481A CN 2012100735297 A CN2012100735297 A CN 2012100735297A CN 201210073529 A CN201210073529 A CN 201210073529A CN 102600481 A CN102600481 A CN 102600481A
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Abstract
The invention discloses an adenovirus (rAdV)/Japanese encephalitis virus (JEV) replicon embedding carrier hog cholera vaccine and an application of the rAdV/ JEV replicon embedding carrier hog cholera vaccine. The recombinant embedded rAdV which carries JEV replicon and stably expresses enhanced green fluorescent protein (EGFP) genes is firstly built. On the basis, hog cholera virus E2 genes are further adopted for replacing the EGFP genes in the embedding carriers, the recombinant embedded virus rAdV-JEV-E2 containing the hog cholera virus E2 genes is built. Indirect immunofluorescence assays prove that the hog cholera virus E2 protein is stably and efficiently expressed in the human embryonic kidney (HEK) 293 cells infected by the recombinant embedded virus rAdV-JEV-E2. Vaccination and challenge assay results prove that the built recombinant embedded virus rAdV-JEV-E2 has a good immune protection effect.
Description
Technical field
The present invention relates to a kind of chimeric vector vaccine and application thereof that carries antigen gene, relate in particular to adenovirus/Japanese encephalitis virus replicon chimeric vector swine Fever Vaccine and application thereof, belong to chimeric vector swine Fever Vaccine field.
Background technology
Adenovirus vector becomes one of the most frequently used viral vector because of it has that host range is extensive, safe, virion is stable, the operation of DNA recombinant technique is reset, is easy to genome lessly, have and efficiently send and advantage such as expression alien gene.Replication defective property people 5 type adenovirus vectors make it can carry big segmental exogenous gene because of having lacked viral dispensable gene district (E1/E3), because it is a replication defective property, people and animal are produced a passing infection again, and are safe.
Rna replicon; It is the self-replicating viral RNA; Be on the basis of picornavirus infection sex clone with other exogenous gene replacement virus structural protein gene, keep its nonstructural protein gene and noncoding region and the expression system that develops; It utilizes the self-replicating characteristic of positive chain RNA virus, can when expressing viral non-structural protein, efficiently express behind the transfectional cell foreign protein (Yu Yunzhou, Yu Weiyuan. flavivirus replicon carrier progress. the biotechnology communication; 2006,17 (2): 228-231).The sub-vaccine of rna replicon has very strong advantage; As: the self-replicating ability is arranged; The proteic expression of self regulatory function of virus does not receive impact cell, viral protein expression amount advantages of higher (Polo JM, Dubensky TW.Virus-based vectors for human vaccine applications.Drug Discovery Today; 2002,7 (13): 719-727.).Weak point is that the sub-carrier of rna replicon can not get in the body effectively.In addition; The flavivirus replicon carrier protein has certain toxic and side effects to the host bacterium, and this possibly be to cause the unsettled principal element of flavivirus replicon carrier (Rice CM, Grakoui A; Galler R; Et al.Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation.New Biol, 1989,1 (3): 285-96.).
There is unstability in banzi virus eDNA in antibacterial; Also there is this situation (Lai CJ in Japanese encephalitis virus (JEV) replicon carrier; Zhao BT, Hori H, et al.Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus.Proc Natl Acad Sci USA; 1991,88 (12): 5139-5143; Sumiyoshi H; Hoke CH, Trent DW, et al.Infectious Japanese encephalitis virus RNA can be synthesized from vitro-ligated cDNA templates.J Virol; 1992,66 (9): 5423-5431.).Do not appear in the newspapers as yet about the research of sending JEV replicon expression vector with adenovirus both at home and abroad at present.
Summary of the invention
One of the object of the invention provides a kind of adenovirus/Japanese encephalitis virus replicon chimeric vector vaccine that carries antigen gene;
Two of the object of the invention provides a kind of said method of carrying the adenovirus/Japanese encephalitis virus replicon chimeric vector vaccine of antigen gene that makes up;
Three of the object of the invention provides a kind of adenovirus/Japanese encephalitis virus replicon chimeric vector swine Fever Vaccine;
Above-mentioned purpose of the present invention realizes through following technical scheme:
A kind of adenovirus/Japanese encephalitis virus replicon chimeric vector vaccine comprises: replication defective property people 5 type adenoviruss, Japanese encephalitis virus replicon and antigen gene to be expressed; Wherein, (that is: be positioned at JEV SA14-14-2 pnca gene group 164-165 position nucleotide) inserts the intron sequences of plasmid pCI before Japanese encephalitis virus replicon SpeI site; Antigen gene to be expressed is between the SpeI and SalI site of JEV replicon carrier.
Wherein, the nucleotides sequence of the intron sequences of said plasmid pCI is classified as shown in the SEQ ID No.1.
" antigen gene to be expressed " of the present invention can be antigen genes such as swine fever virus raq gene, PRRS virus GP5 gene, porcine circovirus 2 type Cap gene or swine influenza virus HA gene; Be preferably the swine fever virus raq gene.
Other a kind of purpose of the present invention provides a kind of method that makes up said adenovirus/Japanese encephalitis virus replicon chimeric vector, may further comprise the steps:
(1) Japanese encephalitis virus (JEV) replicon is inserted in the adenovirus shuttle vector; (2) will be inserted in the JEV replicon from the chimeric intron sequences and the antigen gene to be expressed of pCI carrier, obtain transfer vector; (3) with transforming the competent cell that contains the adenovirus skeleton carrier after the transfer vector linearisation, the gland grain obtains recombinating; (4) with the gained transfectional cell after the linearisation of gland grain of recombinating, go down to posterity, obtain containing the adenovirus/Japanese encephalitis virus replicon chimeric vector vaccine of antigen expressed gene until cytopathy occurring.
Wherein, the adenovirus shuttle vector described in the step (1) comprises pShuttle, pShuttle-CMV, pAdTrack or pAdTrack-CMV etc.; Be preferably pShuttle-CMV;
Before Japanese encephalitis virus replicon SpeI site, insert the intron sequences (SEQ ID No.1) of plasmid pCI in the step (2); In addition, antigen gene to be expressed is inserted between the SpeI and SalI site of JEV replicon carrier.
Cell described in the step (4) is preferably the HEK293 cell.
Employed JEV replicon has kept all non-structural proteins of JEV among the present invention, can in the opposing exogenous virus, to JEV the certain protection effect be arranged also.Adopt adenovirus to send JEV replicon expression system, made full use of the advantage of replicon ability efficiently expressing exogenous gene, solved nucleic acid vaccine again and be not easy to get into the intravital problem of animal.In the inventor's previous work; Obtained the proteic adenovirus of expression EGFP/Japanese encephalitis virus replicon carrier embedded virus; But behind other gene (like swine fever virus raq gene or swine influenza virus HA gene) replacement EGFP gene,, can hold big fragment and the slower DH10B bacterial strain of growth such as using in the conversion process although adopt different corrective measures; And use low temperature, the slow-speed of revolution, low concentration antibiotic-screening; But can't obtain inserting the chimeric vector clone of exogenous gene all the time, can't obtain embedded virus, this possibly be because the unstability of banzi virus cDNA in escherichia coli causes.In order to address this problem; The present invention has attempted in JEV replicon sequence, introducing the method for intron; Avoid the albumen of JEV protein translation generation to the toxic effect of host bacterium; Also attempted introducing sudden change and eliminated methods such as escherichia coli promoter implicit in the JEV sequence, intron sequences is introduced in final discovery can make the JEV replicon carrier become stable, behind swine fever virus raq gene replacement EGFP gene; Be easy to choose positive colony, also correct stably express (table 1) of E2 albumen behind the transfectional cell.
Table 1 is introduced from the influence to the JEV replicon of the chimeric intron of pCI carrier
The present invention has at first obtained to carry the reorganization chimeric adenoviral of JEV replicon, stably express EGFP gene, and recombinant virus is infectious high to the HEK293 cell, and cytopathogenic effect is fast, and it is also very high that fluorescence microscope is observed the EGFP expression down.Wherein, the concrete construction method of the reorganization chimeric adenoviral that carries JEV replicon, stably express EGFP gene of the present invention can be with reference to following technical scheme:
The JEV replicon that contains enhanced green fluorescence protein (EGFP) gene that at first will make up is inserted into after transforming and has in the pShuttle-CMV carrier in SwaI site; To insert from the chimeric intron of pCI carrier in the JEV replicon again; Obtain transfer vector pSh-JEV-In-EGFP; After the SwaI linearisation, transform the BJ5183 competent cell; With the gained gland grain transfection HEK293 cell after the PacI linearisation of recombinating, go down to posterity until cytopathy occurring, obtain containing the reorganization chimeric adenoviral rAdV-JEV-EGFP of the JEV replicon of expressing EGFP.PCR and enzyme action qualification result show, pSh-JEV-In-EGFP passed for 20 generations and still keeps stable in antibacterial, and EGFP albumen is stable in the HEK293 cell that rAdV-JEV-EGFP infects, express efficiently.
The present invention has carried out desk study to adenovirus/Japanese encephalitis virus replicon chimeric vector expression system first; Confirmed to send the feasibility of JEV replicon expression system by adenovirus; The JEV replicon of stable expression of exogenous gene is delivered in the host effectively, sends the new generation vaccine that the JEV replicon is the basis for further research and development with adenovirus and lay the first stone.On this basis, further with the EGFP gene in the swine fever virus raq gene replacement chimeric vector, the Japanese encephalitis virus replicon that utilizes adenovirus to send is carrier in the present invention, has made up the reorganization chimeric adenoviral rAdV-JEV-E2 that expresses the swine fever virus raq gene.
The present invention carries out preservation with the preservation mechanism that constructed reorganization chimeric adenoviral rAdV-JEV-E2 is submitted to patent approval, and its microbial preservation number is: CGMCC No.5825; The classification name is: send the recombinant adenovirus of expressing the proteic Japanese encephalitis virus replicon of swine fever virus E2; The preservation time is: on February 29th, 2012; Depositary institution is: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The present invention adopts indirect immunofluorescence assay conclusive evidence swine fever virus E2 albumen in the metainfective HEK293 cell of recombinant virus rAdV-JEV-E2, to be stablized, express efficiently; Rabbit immunity challenge test is the result show; 2 weeks can detect the generation of swine fever specific antibody after the rAdV-JEV-E2 immunity; Reaching peak value (part rabbit antibody blocking rate reaches more than 80%) 5 weeks after the immunity; The same with the rabbit that inoculates the C strain behind the counteracting toxic substances all the appearance typing is hot, and the whole appearance typings behind counteracting toxic substances of the contrast rabbit of inoculation rAdV-JEV-EGFP are hot, and this explanation rAdV-JEV-E2 has better immunogenicity.Utilizing the hog cholera lapinised virus strain that immunizing rabbit is carried out counteracting toxic substances is classical swine Fever Vaccine Evaluation Strategy; Therefore the present invention chimeric adenoviral rAdV-JEV-E2 that recombinates has good immunoprotective effect; Can be used as swine Fever Vaccine and use, this shows further that also the constructed adenovirus of the present invention/Japanese encephalitis virus replicon chimeric vector vaccine is a novel vaccine carrier that application potential is arranged very much.
Description of drawings
Fig. 1 introduces the intron sequences structural representation; P
CMV: the CMV promoter; 5 of 5 ' NCR and 3 ' NCR:JEV ' and 3 ' noncoding region; Preceding 23 aminoacid of C23:JEV C albumen; Intron: from the chimeric intron of pCI carrier; EGFP: green fluorescent protein; E2: swine fever virus E2 albumen; Preceding 25 amino acids of E25:JEVE albumen; The NS1-5:JEV non-structural protein; HDVRz: hepatitis D virus ribozyme; PolyA:SV40 polyA signal.
Reach the enzyme action evaluation of 20 generations in Fig. 2 pSh-JEV-In-EGFP antibacterial; 1:DL15, the 000DNA molecular weight standard; 2:BglII; 3:EcoRI; 4:BglII+EcoRI; 5:DL2, the 000DNA molecular weight standard.
Fig. 3 EGFP infects the expression in the HEK293 cell at reorganization chimeric adenoviral rAdV-JEV-EGFP; The HEK293 cell that A:rAdV-JEV-EGFP infects; B: normal HEK293 cell.
Fig. 4 detects reorganization chimeric adenoviral rAdV-JEV-E2 with indirect immunofluorescence assay and infects E2 protein expression in the HEK293 cell; The HEK293 cell that A:rAdV-E2 infects; The HEK293 cell that B:rAdV-JEV-E2 infects; C: normal HEK293 cell.
The swine fever specific antibody level that produces after the immunity of Fig. 5 rabbit.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
Structure and the evaluation of embodiment 1 reorganization chimeric adenoviral rAdV-JEV-EGFP and rAdV-JEV-E2
1 material method
1.1 cell, bacterial strain, plasmid vector
The HEK293 cell is preserved by Harbin veterinary institute Vet Biotechnology National Key Laboratory.The AdEasy-1 competence bacteria of adenovirus shuttle vector pShuttle-CMV and gland-containing virus skeleton carrier is available from Stratagene company.The JEV replicon pOK-JEV-EGFP that carries green fluorescent protein (EGFP) gene according to the method for bibliographical information make up (foundation of the bright .JEV replicon carrier of old filial piety system and in the application of PRRS vaccine. Guangzhou: Agricultural University Of South China's veterinary college, 2010).
1.2 the transformation of transfer vector pShuttle-CMV
According to the synthetic a pair of 5 ' end of pShuttle-CMV sequential design 9 primer P1, P2 that base is identical are arranged, and introduce restriction enzyme site KpnI, EcoRI, NotI.Primer sequence is (underscore is for introducing restriction enzyme site) as follows:
P1：5′-TCA
GAATTCGGTACCCGGATCTGGGCGTGGTTAAG-3′(SEQ?ID?No.2)
P2：5′-TCA
GAATTCGCGGCCGCACAGTATTACGCGCTATGAGTAACA-3′(SEQID?No.3)
With pShuttle-CMV is template; Carry out pcr amplification with
HS archaeal dna polymerase; Obtain the cyclic plasmid (leaving out CMV-MCS-SV40polyA) of 6500bp size, behind DNA test kit purification, add the DpnI digestion with restriction enzyme and fall plasmid template; Again through DNA purification kit purification; Transform DH5 α, extract plasmid enzyme restriction and identify recombiant plasmid called after pShuttle.
According near the known array the pShuttle PmeI, the synthetic a pair of primer P3/P4 of design, the upstream and downstream primer is introduced identical restriction enzyme site SwaI, so that PmeI is sported SwaI.Primer sequence is (underscore is a restriction enzyme site) as follows.
P3：5′-A
ATTTAAATGAATTCAATAGCTTGT-3′(SFQ?ID?No.4)
P4：5′-TTC
ATTTAAATTCCCTCTCAAGTCT-3′(SEQ?ID?No.5)
With plasmid pShuttle is template; Carry out pcr amplification with
HS archaeal dna polymerase; Amplified fragments is not for the plasmid full length sequence in PmeI site; Purified pcr product; Fully the back is from connecting with the SwaI digestion with restriction enzyme, and selecting can be by SwaI enzyme action and can not be by the clone of PmeI enzyme action, called after pShuttle-SwaI.
1.3pSh-JEV-EGFP the structure of shuttle vector
Plasmid pOK-JEV-EGFP is used the NotI/KpnI double digestion, and glue reclaims CMV to the SV40polyA fragment, is inserted between the NotI and KpnI of pShuttle-SwaI, obtains pSh-JEV-EGFP.
1.4 introducing intron sequences
For increasing the stability of JEV replicon, before replicon carrier SpeI site, introduce plasmid pCI (Promega company) intron sequences (Fig. 1), to stop the protein expression of the implicit antibacterial promoter control of replicon through homologous recombination.Primer sequence is (underscore is the homology arm sequence) as follows:
PJEV-IN-EGFP-S：
5′-
GGGCTATCAATATGCTGAAACGCGGCCTACCCCGCGTAAGTATCAAGGTTACAAG-3′(SEQ?ID?No.6)
PJEV-IN-EGFP-R：
5′-
AACAGCTCCTCGCCCTTGCTCACCATACTAGTCTGTGGAGAGAAAGGCAAAG-3′(SEQ?ID?No.7)
With pCI is the intron sequences that template amplification carries JEV carrier homologous sequence; With the PCR product and through the linearizing plasmid pSh-JEV-EGFP of SpeI cotransformation DH5 α competence bacteria; Utilize the recombinase in the antibacterial to carry out homologous recombination; Extract plasmid and identify positive plasmid called after pSh-JEV-In-EGFP through PCR.PSh-JEV-In-EGFP is gone down to posterity in antibacterial 20 times, identify, can cut out and expect the band that size conforms to (Fig. 2) through BglII, EcoRI single endonuclease digestion and BglII and EcoRI double digestion.
1.5 the structure of shuttle vector pSh-JEV-In-E2
According to pSC-E2 sequence (Yuan Sun; Qiaofen Qi; Bingbing Liang, et al. express the structure of the proteic reorganization chimeric adenoviral of swine fever virus E2 and in the intravital immunogenicity analysis of rabbit. biological engineering journal, 2008; 24 (10): 1734-1739.), the raq gene of the method amplification swine fever virus through PCR.The design of primers of E2 (underscore is for introducing restriction enzyme site):
PE2S：
5′-TAGGTACCTCTAGAGCCACCATGGTATTAAGAGGACAG-3′(SEQ?ID?No.8)
PE2R：5′-CGAAGCTTGTCGACTTAACCAGCGGCGAGCTGTTC-3′(SEQ?ID?No.9)
Primer is introduced XbaI and SalI restriction enzyme site respectively.Use the XbaI/SalI double digestion, glue reclaims the E2 fragment, is inserted between the SpeI/SalI of pSh-JEV-In-EGFP, obtains pSh-JEV-In-E2.
1.6 the structure of reorganization gland grain pAdV-JEV-In-EGFP and pAdV-JEV-In-E2
Plasmid pSh-JEV-In-EGFP and pSh-JEV-In-E2 are used the SwaI linearisation respectively, and electricity is converted in the BJ5183 competence antibacterial of gland-containing virus skeleton carrier AdEasy-1 and carries out homologous recombination behind the DNA purification kit purification.Through the kalamycin resistance screening, extract plasmid and identify that with the PacI enzyme action gland grain pAdV-JEV-In-EGFP and pAdV-JEV-In-E2 obtain recombinating.
1.7 transfection
Extract pAdV-JEV-In-EGFP and pAdV-JEV-In-E2; After using the PacI linearisation respectively; Reuse DNA purification kit carries out purification; With the HEK293 cell monolayer in concentration difference transfection to six orifice plate in 6 μ g DNA/ holes, wherein a hole is left intact as negative control with Lipofectamine 2000 transfection reagent by specifications.
1.8 the purification and the evaluation of reorganization chimeric adenoviral
Changed in per two days after the transfection and once keep liquid, up to the 8th day, collect the culture multigelation 3 times, the centrifugal 5min of 5000r/min collects supernatant in-20 ℃ of preservations.And in six orifice plates, cultivate the HEK293 cell, when cell reaches 80%-90%, discard culture fluid; Inoculation 1ml frozen culture supernatant behind the absorption 2h, adds and keeps liquid and cultivate to 2ml; And behind 3d as above method go down to posterity once; Observe luciferase expression and cytopathogenic effect (CPE) situation every day, when transfection pAdV-JEV-In-EGFP group has a large amount of luciferase expressions and a large amount of pathological changes of cell and transfection pAdV-JEV-In-E2 group a large amount of pathological changes to occur, get 2 μ l virus liquid in fresh cell monolayer; Observe the luciferase expression situation; The difference collecting cell extracts the total DNA of cell behind the 48h, is the PCR positive control with gland grain pAdV-JEV-In-EGFP and pAdV-JEV-In-E2, extracts normal HEK293 cell DNA in addition as negative control.With identical primer amplification, 1% sepharose electrophoresis is analyzed, and identifies correct recombinant virus called after rAdV-JEV-EGFP and rAdV-JEV-E2 respectively.
With swine fever virus E2 protein monoclonal antibody 5B1 reorganization chimeric adenoviral rAdV-JEV-E2 is infected the HEK293 cell simultaneously and carry out indirect immunofluorescence assay.Method is following: the HEK293 cell is laid in 6 orifice plates, during to 80% monolayer, inoculates 2 μ l rAdV-JEV-E2 when long, the HEK293 cell that behind the 48h rAdV-JEV-E2 is infected is with the fixing 20min of 4% paraformaldehyde room temperature; The 1%BSA room temperature is sealed 30min, adds the monoclonal antibody 5B1 of anti-CSFV, 37 ℃ of effect 2h; After the PBS washing, add 37 ℃ of effects of fluorescent labeling sheep anti-mouse igg 30min, after the PBS washing; Dry up, add buffering glycerol, observe down in fluorescence microscope.
Behind reorganization gland grain pAdV-JEV-EGFP transfection HEK293 cell; Instantaneous luciferase expression is arranged; Do not see behind the 8d that CPE forms; In blind passage to 4-5 generation, pathological changes (cell rounding, shinny, to the periphery diffusion) appears in cell, and fluorescence microscope can be observed EGFP albumen great expression (Fig. 3) in the HEK293 cell that rAdV-JEV-EGFP infects down.
Reorganization chimeric adenoviral rAdV-JEV-E2 begins to occur pathological changes when reaching for the 5th generation, and complete in the 48h pathological changes, and indirect immunofluorescence assay can detect the expression (Fig. 4) of swine fever virus E2 albumen in the HEK293 cell that infects.
Test Example 1 animal immune and challenge test
Choose healthy 13 of White Rabbits of screech owl (available from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center) in 14 ages in week, be divided into three groups of A, B, C at random, 4 of A groups, 5 of B groups, 4 of C groups.A, B organize respectively through back leg intramuscular inoculation 1mL 10
5TCID
50Reorganization chimeric adenoviral rAdV-JEV-EGFP, rAdV-JEV-E2, C group is through ear vein injection 100RID
50Hog cholera lapinised virus vaccine C strain (the C strain is available from Harbin Weike Biologic Technology Ltd.).Immunity back 7w uses 100RID
50Through the ear vein route of inoculation all test rabbits are attacked, whenever behind the counteracting toxic substances surveyed rectal temperature at a distance from 6h, METHOD FOR CONTINUOUS DETERMINATION 3d confirms whether rabbit exists the typing thermal response, and body temperature is higher more than 1 ℃ and keep 18h and be judged to the typing thermal response than basal temperature.3d behind the counteracting toxic substances; Cut open and kill all test rabbits; Gather spleen, use the fluorescent quantitative RT-PCR method (Zhao JJ, the Cheng D that have reported; Li Na; Et al.Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of Classical swine fever virus.Vet Microbiol, 2008,126 (1/3): 1-10) detect wherein rabbitization attenuated vaccine viral RNA content.
Immunity back 7w; After rabbits attacked to all tests with the C strain, thermal response did not all appear in rAdV-JEV-E2 and all rabbit of C strain immune group, and typing thermal response (table 2) has all appearred in 4 rabbit in the rAdV-JEV-EGFP matched group; Fluorescence quantitative RT-RCR detects and shows; Virus-free RNA in the rAdV-JEV-E2 immunize rabbit spleen, and all have a large amount of viral RNAs in the contrast rabbit spleen, be up to 10
4Copy/μ L (table 3) shows that rAdV-JEV-E2 can induce complete immunoprotection.
Table 2 with C strain counteracting toxic substances after each immune group rabbit produce the testing result of typing thermal response
Viral RNA detection by quantitative in the spleen behind the table 3 immunizing rabbit counteracting toxic substances
Annotate :-detect less than.
Before the immunity and after the immunity weekly with counteracting toxic substances after the 3rd day to all immunizing rabbits through ear vein blood sampling; Collect the blocking-up ELISA test kit of using based on CSFV E2 albumen neutralizing monoclonal antibody behind the serum (available from IDEXX company) and detect hog cholera antibody, the concrete operation method by specification carries out.
The rAdV-JEV-E2 immune group is in beginning to produce swine fever specific antibody (the blocking-up rate reaches and is judged to be antibody positive more than 40%) the 2nd week after the immunity, and antibody horizontal raises gradually subsequently; C strain immune group is at the hog cholera antibody that begins to produce higher titre the 1st week, and rAdV-JEV-E2 4 all antibody after immunity all change sun, and rAdV-JEV-EGFP immunity matched group does not produce swine fever specific antibody (Fig. 5) all the time.
< 110>Harbin Veterinary Medicine Inst., China Academy of Agriculture
< 120>adenovirus/Japanese encephalitis virus replicon chimeric vector swine Fever Vaccine and application thereof
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Claims (10)
1. adenovirus/Japanese encephalitis virus replicon chimeric vector vaccine is characterized in that, comprising: replication defective property people 5 type adenoviruss, Japanese encephalitis virus replicon and antigen gene to be expressed.
2. according to the described adenovirus of claim 1/Japanese encephalitis virus replicon chimeric vector vaccine, it is characterized in that: at the Japanese encephalitis virus replicon
SpeInsert the intron sequences of plasmid pCI before the I site; The nucleotides sequence of the intron sequences of said plasmid pCI is classified as shown in the SEQ ID No.1.
3. according to the described adenovirus of claim 1/Japanese encephalitis virus replicon chimeric vector vaccine, it is characterized in that: antigen gene to be expressed is positioned at the JEV replicon carrier
SpeI with
SalBetween the I site.
4. according to any one described adenovirus of claim 1-3/Japanese encephalitis virus replicon chimeric vector vaccine, it is characterized in that: described antigen gene to be expressed is swine fever virus raq gene, PRRS virus GP5 gene, porcine circovirus 2 type Cap gene or swine influenza virus HA gene.
5. any one described adenovirus of claim 1-3/Japanese encephalitis virus replicon chimeric vector vaccine is in the purposes of preparation in the animal vaccine.
6. method that makes up any one said adenovirus of 1-3/Japanese encephalitis virus replicon chimeric vector vaccine may further comprise the steps:
(1) the Japanese encephalitis virus replicon is inserted in the adenovirus shuttle vector; (2) will be inserted in the Japanese encephalitis virus replicon from the chimeric intron and the antigen gene to be expressed of pCI carrier, obtain recombinant transfer vector; (3) with transforming the competent cell that contains the adenovirus skeleton carrier after the transfer vector linearisation, the gland grain obtains recombinating; (4) transfectional cell after the linearisation of gland grain of will recombinating goes down to posterity until cytopathy occurring, obtains containing the adenovirus/Japanese encephalitis virus replicon chimeric vector vaccine of antigen expressed gene.
7. according to the described method of claim 6, it is characterized in that: the adenovirus shuttle vector described in the step (1) comprises pShuttle, pShuttle-CMV, pAdTrack or pAdTrack-CMV; Be preferably pShuttle-CMV.
8. according to the described method of claim 6, it is characterized in that: in the step (2) at the Japanese encephalitis virus replicon
SpeInsert the intron sequences of plasmid pCI before the I site; Antigen gene to be expressed is inserted into the JEV replicon carrier
SpeI with
SalBetween the I site.
9. adenovirus/Japanese encephalitis virus replicon chimeric vector swine Fever Vaccine, its microbial preservation number are: CGMCC.5825.
10. the described adenovirus of claim 9/Japanese encephalitis virus replicon chimeric vector swine Fever Vaccine prevents Japanese encephalitis virus or/and the purposes in the vaccine of swine fever virus in preparation.
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| CN2012100735297A CN102600481A (en) | 2012-03-20 | 2012-03-20 | Adenovirus (rAdV)/Japanese encephalitis virus (JEV) replicon embedding carrier hog cholera vaccine and application of rAdV/ JEV replicon embedding carrier hog cholera vaccine |
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| CN103088049A (en) * | 2012-12-26 | 2013-05-08 | 中国人民解放军军事医学科学院生物工程研究所 | DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain, as well as construction method and application thereof |
| CN108904793A (en) * | 2018-07-29 | 2018-11-30 | 首都医科大学附属北京友谊医院 | The DNA vaccination and its construction method for preventing Japanese Type-B encephalitis |
| CN110088273A (en) * | 2016-11-28 | 2019-08-02 | 梅迪根股份有限公司 | For the vaccine of infectious diseases caused by just strand rna virus |
| CN111454989A (en) * | 2020-04-04 | 2020-07-28 | 中国农业科学院兰州兽医研究所 | Chimeric gene type I encephalitis B virus-like particle vaccine and preparation method and application thereof |
| CN116716322A (en) * | 2023-05-12 | 2023-09-08 | 中山大学 | DENV-3 full-length infectious clone and construction method and application thereof |
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| CN103088049A (en) * | 2012-12-26 | 2013-05-08 | 中国人民解放军军事医学科学院生物工程研究所 | DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain, as well as construction method and application thereof |
| CN110088273A (en) * | 2016-11-28 | 2019-08-02 | 梅迪根股份有限公司 | For the vaccine of infectious diseases caused by just strand rna virus |
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| CN108904793A (en) * | 2018-07-29 | 2018-11-30 | 首都医科大学附属北京友谊医院 | The DNA vaccination and its construction method for preventing Japanese Type-B encephalitis |
| CN111454989A (en) * | 2020-04-04 | 2020-07-28 | 中国农业科学院兰州兽医研究所 | Chimeric gene type I encephalitis B virus-like particle vaccine and preparation method and application thereof |
| CN111454989B (en) * | 2020-04-04 | 2022-03-08 | 中国农业科学院兰州兽医研究所 | Chimeric gene type I encephalitis B virus-like particle vaccine and preparation method and application thereof |
| CN116716322A (en) * | 2023-05-12 | 2023-09-08 | 中山大学 | DENV-3 full-length infectious clone and construction method and application thereof |
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