CN103540604A - Building method of deoxyribonucleic acid (DNA) replicor vaccine vector of swine Japanese encephalitis virus and use thereof - Google Patents
Building method of deoxyribonucleic acid (DNA) replicor vaccine vector of swine Japanese encephalitis virus and use thereof Download PDFInfo
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- CN103540604A CN103540604A CN201310502189.XA CN201310502189A CN103540604A CN 103540604 A CN103540604 A CN 103540604A CN 201310502189 A CN201310502189 A CN 201310502189A CN 103540604 A CN103540604 A CN 103540604A
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Abstract
The invention discloses a building method of a deoxyribonucleic acid (DNA) replicor vaccine vector of a swine Japanese encephalitis virus and use thereof, and belongs to the technical field of biological medicines. Virus structural protein genes prM and E are removed while replicor expression vectors PAC-JEV of all non-structural protein are reserved on the basis of infectious clone of built Japanese encephalitis virus (JEV) vaccine strain SA14-14-2. A replicor PAC-JEV target gene is recombined to transfect BHK-21 after the target gene is inserted into the downstream of a C gene of a structural protein gene. The DNA replicor vaccine vector of the swine Japanese encephalitis virus can be applied to research of a functional relationship of the JEV gene and the protein, and a vaccine based on the replicor vector.
Description
Technical field
The invention belongs to biological medicine technology field, be specially construction process of a boar Japanese B encephalitis virus amplicon dna vaccine and uses thereof.
Background technology
Virus replication refers to remove virus structural protein gene and retains to the viral subgenomic that copies relevant nonstructural protein gene because of group, and this subgene group has the target protein that self-replicating accurate translation non-structural protein bletilla insert.Therefore virus replication is not only research virogene structure and function technology platform, is also the novel means of expressing foreign protein and the sub-vaccine of research and development New Replication.The replicon of the RNA viruses such as existing Alphavirus, picornavirus, flavivirus, coronavirus successfully constructs report; what wherein research was the most ripe is Alphavirus replicon; succeeded in developing the commercialization Alphavirus replicon product as carrier for expression of eukaryon; the Alphavirus replicon of take is avoided virus infection as basic new generation vaccine is also proved to be to watch for animals simultaneously; yet Alphavirus replicon cytotoxicity causes by force apoptosis very soon, can not continuous expression foreign protein.Flavivirus replicon has but overcome above-mentioned shortcoming, and flavivirus replicon cytotoxicity is very little, can be in the tens of skies of time multiplexed cell system; The fidelity of the RNA polysaccharase of flavivirus high and while guaranteeing flavivirus replicon as vaccine carrier immunogenicity more stable; The RNA of flavivirus replicon is less than 10000nt and is convenient to genetic manipulation, so the research of flavivirus replicon has scientific research value.
Encephalitis b virus (Japanese encephalitis virus, JEV) be a kind of important flaviviridae (Flaviviridae family) Flavivirus (Flavivirus) member, belong to virus together and also comprise yellow fever virus (Yellow fever virus, YEV), tick-brone encephalitis virus (Tick-borne encephalitis virus, TBEV) and dengue fever virus (Dengue virus, DENV) etc.JEV virus particle diameter 50nm, viral genome sub-thread positive chain RNA, is about 11kb, containing a large open reading frame encode structural protein C, prM viral, envelope protein E and 7 non-structural protein NS 1s, NS2A, NS2B, NS3, NS4A, NS4B, NS5.JEV genome 5 ' end has I type cap sequence (m7GpppAm p), and 3 ' without poly A tail.Genome 5 ' and 3 ' terminal nucleotide all can the conservative secondary structures of height of formation, participate in copying of virus, and the replicative enzyme of NS3, NS5 genes encoding copies and translates viral genome is necessary.
Summary of the invention
For the above-mentioned problems in the prior art, the object of the invention is to design the technical scheme that construction process of a boar Japanese B encephalitis virus amplicon dna vaccine and uses thereof is provided, its construction process is simple, the vaccine stable performance of acquisition, cost is low, and effect is remarkable.
The construction process of a described boar Japanese B encephalitis virus amplicon dna vaccine carrier, is characterized in that comprising the following steps:
1) by the gene fragment plasmid pK-JEV that contains JEV vaccine strain SA14-14-2, the low copy plasmid pACYC-177 of take is skeleton, introduce new restriction enzyme site (Hind III-EcoR I-Kpn I-Sal I-Xba I-Xho I), CMV promotor is inserted between new restriction enzyme site Hind III-EcoR I of introducing; Respectively JEV structural protein C gene, FMDV 2A are inserted between EcoR I and Kpn I restriction enzyme site afterwards, be called pAC-CMV-CF2A plasmid; Take pK-JEV as template again, with NS I-F and NS I-R, NS II-F and NS II H-R1/R2/R3 increase respectively JEV Nonstructural Protein NS I and NS II+HDVRz, NS I is stored in to-20 ℃, standby;
2) utilizing Xba I and Xho I that NS II is connected to pBluescript SK2 (+) goes up and is called pBlue-NS II+HDVRz, take Nhe I and Xho I is connected to pBlue-NS II+HDVRz by polyA as restriction enzyme site, connect Product Identification and correctly by Xba I and Xho I double digestion, be connected to pAC-CMV-CF2A above, be referred to as pAC-exJEV;
3) finally utilize Sal I and Xba I that NS I is connected to pAC-exJEV upper, final replicon carrier is called to pAC-JEV, obtain amplicon dna vaccine.
The construction process of a described boar Japanese B encephalitis virus amplicon dna vaccine, the JEV vaccine strain SA14-14-2 infections clone that it is characterized in that building is basis, removes virus structural protein gene prM and E and retains the replicon expression vector pAC-JEV of whole Nonstructural Proteins.
The construction process of a described boar Japanese B encephalitis virus amplicon dna vaccine carrier, is characterized in that after EGFP or goal gene are inserted in structural protein gene C gene downstream the sub-PAC-JEV-EGFP of this recombinant replication or goal gene transfection BHK-21.
The described application of boar Japanese B encephalitis virus amplicon dna vaccine carrier on JEV gene and protein functional relationship and the vaccine research based on replicon carrier.
The construction process of an above-mentioned boar Japanese B encephalitis virus amplicon dna vaccine carrier, its construction process is simple, the vaccine stable performance of acquisition, cost is low, and effect is remarkable; This replicon expression vector can, for the emic research of JEV gene and protein, be applied to the research based on replicon carrier vaccines.
The percentage composition relating in present specification unless otherwise indicated, other the weight percentage that is pure substance.
Accompanying drawing explanation
Fig. 1 is replicon and related vector structural representation;
Fig. 2 is that replicon pAC-JEV-EGFP is in the expression of BHK-21 cell;
In Fig. 2: A.12h; B.48h; C.8d; D. Negative control (BHK-21 cells transfection with pAC-JEV-EGFP).
Embodiment
Now, in conjunction with example of the present invention, further illustrate beneficial effect of the present invention.
Embodiment
By the gene fragment plasmid pK-JEV that contains JEV vaccine strain SA14-14-2, the low copy plasmid pACYC-177 of take is skeleton, introduce new restriction enzyme site (Hind III-EcoR I-Kpn I-Sal I-Xba I-Xho I), CMV promotor is inserted between new restriction enzyme site Hind III-EcoR I of introducing, respectively JEV structural protein C gene, FMDV 2A are inserted between EcoR I and Kpn I restriction enzyme site afterwards, be called pAC-CMV-CF2A plasmid, take pK-JEV as template again, with NS I-F and NS I-R, NS II-F and NS II H-R1/R2/R3 increase respectively JEV Nonstructural Protein NS I and NS II+HDVRz, NS I is stored in to-20 ℃, and then utilize Xba I and Xho I that NS II is connected to pBluescript SK2 (+) and go up and be called pBlue-NS II+HDVRz, take Nhe I and Xho I is connected to pBlue-NS II+HDVRz by polyA as restriction enzyme site, connecting Product Identification is correctly connected on pAC-CMV-CF2A by Xba I and Xho I double digestion, be referred to as pAC-exJEV, finally utilize Sal I and Xba I that NS I is connected on pAC-exJEV, final replicon carrier is called to pAC-JEV.Again by the pAC-JEV obtaining with reference to Lipofectamine2000 specification sheets method transfection BHK-21 cell, obtain amplicon dna vaccine.
By test, further illustrate useful test-results of the present invention below.
Test one:
Test materials: the gene fragment plasmid pK-JEV that contains JEV vaccine strain SA14-14-2; Plasmid pBluescript SK2 (+), pcDNA3.1, pEGFP-N1, how anti-low copy plasmid pACYC-177, the anti-JEV of mouse be by Zhejiang A & F University's Animal Lab., provides and prepare.
1. contain the structure of the JEV replicon carrier pAC-JEV of CMV promotor
JEV replicon carrier pAC-JEV structure is as Fig. 1, and the low copy plasmid pACYC-177 of take is skeleton, introduces new restriction enzyme site (Hind III-EcoR I-Kpn I-Sal I-Xba I-Xho I), and CMV promotor is inserted to the new restriction enzyme site of introducing
hind III-
ecobetween R I, respectively JEV structural protein C gene, FMDV 2A are inserted afterwards
ecor I and
kpnbetween I restriction enzyme site, naming this plasmid is pAC-CMV-CF2A, take pK-JEV as template again, with NS I-F and NS I-R, NS II-F and NS II H-R1/R2/R3 increase respectively JEV Nonstructural Protein NS I and NS II+HDVRz(the two comprise whole nonstructural protein genes), first NS I is stored in-20 ℃ standby, utilize Xba I and Xho I that NS II is connected to the upper also called after pBlue-NS II+HDVRz of pBluescript SK2 (+), take Nhe I and Xho I is connected to pBlue-NS II+HDVRz by polyA as restriction enzyme site, connecting Product Identification is correctly connected on pAC-CMV-CF2A by Xba I and Xho I double digestion, by its called after pAC-exJEV, finally utilize Sal I and Xba I that NS I is connected on pAC-exJEV, by final replicon carrier called after pAC-JEV.
2. transfection BHK-21 cell
Carry greatly pAC-JEV plasmid, by plasmid transfection BHK-21 cell, concrete grammar is with reference to Lipofectamine2000 specification sheets.By square formation, test the optimum ratio of determining plasmid and liposome consumption, afterwards plasmid is dyed to 24 orifice plate individual layer BHK-21 cells, with the negative contrast of pACYC-177 empty plasmid.
3. contain the structure of EGFP reporter gene replicon
Take pEGFP-N1 as template, synthetic two the primer EGFP-F/R (F:5 of design
,gTAGGTACCGTGAGCAAGGG3
,; R:5
,tATGTCGACCTTGTACAGCTCGT3
,), pcr amplification obtains EGFP gene, utilize Kpn I and Sal I double digestion to be connected with pAC-JEV and called after pAC-JEV-EGFP, after sequence verification, Lipofectamine2000 transfection BHK-21, simultaneously with the negative contrast of empty carrier pACYC-177, and 12h starts after transfection, every 24h fluorescence microscopy Microscopic observation change in fluorescence again.
Experimental result:
1. contain the structure of the JEV replicon carrier pAC-JEV of CMV promotor
The elements such as CMV promotor, HDVRz, PolyA terminator are introduced in plasmid pACYC-177, on the basis of the JEV infections clone building in this laboratory, remove JEV structural protein prM and E, built the JEV replicon carrier pAC-JEV(that retains whole Nonstructural Proteins and seen Fig. 1).For convenience of the contrast of follow-up test, then by PCR method, lack the plasmid pAC-JEV-△ NS5(that NS5 carboxyl terminal portion gene sequence builds and see Fig. 1).
2. contain the structure of reporter gene EGFP replicon
Reporter gene EGFP is inserted into replicon carrier pAC-JEV and is built into replicon pAC-JEV-EGFP, after identifying correctly, after Lipofectamine2000 transfection BHK-21, every 12h fluorescence microscopy Microscopic observation.Result shows: fluorescence appear in 12h, the sustainable 8d of fluorescence left and right, during find the some positive cell rounding phenomena of mortality.
Claims (4)
1. the construction process of a boar Japanese B encephalitis virus amplicon dna vaccine carrier, is characterized in that comprising the following steps:
1) by the gene fragment plasmid pK-JEV that contains JEV vaccine strain SA14-14-2, the low copy plasmid pACYC-177 of take is skeleton, introduce new restriction enzyme site (Hind III-EcoR I-Kpn I-Sal I-Xba I-Xho I), CMV promotor is inserted between new restriction enzyme site Hind III-EcoR I of introducing; Respectively JEV structural protein C gene, FMDV 2A are inserted between EcoR I and Kpn I restriction enzyme site afterwards, be called pAC-CMV-CF2A plasmid; Take pK-JEV as template again, with NS I-F and NS I-R, NS II-F and NS II H-R1/R2/R3 increase respectively JEV Nonstructural Protein NS I and NS II+HDVRz, NS I is stored in to-20 ℃, standby;
2) utilizing Xba I and Xho I that NS II is connected to pBluescript SK2 (+) goes up and is called pBlue-NS II+HDVRz, take Nhe I and Xho I is connected to pBlue-NS II+HDVRz by polyA as restriction enzyme site, connect Product Identification and correctly by Xba I and Xho I double digestion, be connected to pAC-CMV-CF2A above, be referred to as pAC-exJEV;
3) finally utilize Sal I and Xba I that NS I is connected to pAC-exJEV upper, final replicon carrier is called to pAC-JEV, obtain amplicon dna vaccine.
2. the construction process of a boar Japanese B encephalitis virus amplicon dna vaccine as claimed in claim 1, the JEV vaccine strain SA14-14-2 infections clone that it is characterized in that building is basis, removes virus structural protein gene prM and E and retains the replicon expression vector pAC-JEV of whole Nonstructural Proteins.
3. the construction process of a boar Japanese B encephalitis virus amplicon dna vaccine carrier as claimed in claim 1, is characterized in that after EGFP or goal gene are inserted in structural protein gene C gene downstream the sub-PAC-JEV-EGFP of this recombinant replication or goal gene transfection BHK-21.
4. the application of boar Japanese B encephalitis virus amplicon dna vaccine carrier on JEV gene and protein functional relationship and the vaccine research based on replicon carrier as claimed in claim 1.
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| CN102250950A (en) * | 2011-05-25 | 2011-11-23 | 华南农业大学 | DNA level-based encephalitis B virus replicon vector system, and construction method and application thereof |
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| CN102250950A (en) * | 2011-05-25 | 2011-11-23 | 华南农业大学 | DNA level-based encephalitis B virus replicon vector system, and construction method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 孙鹤: "连接肽在多基因转化中的应用", 《生物技术进展》 * |
| 王晓杜: "猪日本乙型脑炎检测技术研究进展", 《中国畜牧兽医》 * |
| 黄莺: "日本脑炎病毒(JEV) 复制子表达载体的构建及其鉴定", 《病毒学报》 * |
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Application publication date: 20140129 |