Construction And Identification Of Plasmid-based Replicon Vector System Derived From XJ-160 Virus

ObjectiveXJ-160 virus was isolated from a pool of Anopheles mosquitoes collected in Xinjiang China in 1990. This virus belongs to Sindbis virus. Currently our group has completed the virus genome sequencing, whole genome infectious cDNA clone (pBR-XJ160) construction and RNA-based replicon vector system set up. However, RNA-based replicon vector promoter is prokaryotic promoter, the application requires in vitro transcription, the operation tedious, compared with the RNA-based replicon vector plasmid-based replicon vector can start the transcription through the eukaryotic promoter (eg CMV).It can be directly transfected into cells to achieve a high level of exogenous gene expression without in vitro transcription. Therefore,we construct XJ-160 virus plasmid-based replicon vector system based on pBR-XJ160.Methods1. Construct XJ-160 virus plasmid-based replicon vector, on the base of pBR-XJ160 we divid the virus non-structural gene sequence into three fragments to amplify:XJ1(1-2527nt), XJ2(2527-5161nt), XJ3(5161-7562nt). clone the gene into eukaryotic expression vector pVAX1 CMV promoter downstream, and with multiple cloning sites replace the virus structural gene sequences constructed XJ-160 virus plasmid-based replicon vector pVa-XJ. Using a single restriction enzyme is NheI/BamHI, BamHI/EcoRI, EcoRI/NotI respectively. With multiple cloning sites (multiple clone sites, MCS) sequence replace the the virus structure gene, MCS is composed by NotI, PvuI, FseI, PacI and AscI five single restriction enzyme sequence.2. Construct helper plasmid, helper plasmid for replicon vector can provide virus structural protein trans to package the replication defective virus particles, and ultimately improve the efficiency of vector packaging.Therefor, on the base of pBR-XJ160 we amplified virus nucleoprotein gene C (7563-8354nt) and the envelope glycoprotein gene E (8355-11297nt) respectively,and clone it to pVAX1 CMV promoter downstream respectively to construct two helper plasmids pVa-C and pVa-E, the single restriction enzyme were:EcoRI/XhoIand BamHI/XhoI.3. Function identification of replicon vector system, we inserted green fluorescent protein (EGFP) reporter gene and Gaussia Luciferase(GLUC) report genes into replicon vector multiple cloning site to constructe containing the reporter gene expression plasmid pVaXJ-EGFP and pVaXJ-GLUC, the single restriction enzyme were:NotI/AscI and FseI/ AscI. Co-transfected the reporter gene expression plasmid and helper plasmid into BHK-21 cells, observe green fluorescent protein expression and detect luciferase activity to qualitify and quantitify the functional identification of replicon vector system.Results1.We constructed XJ-160 virus vector-based replicon vector pVa-XJ,with a single restriction enzyme NotI, PvuI, FseI, PacI, and AscI digesting can obtain about 10.5kb liner vector fragment; with NheI/BamHI, BamHI/EcoRI and EcoRI/NotI double digesting can obtain about 2.5kb and 8kb of linear fragments, gene sequencing results also show that the sequence of the replicon vector are correct.2.We constructed two helper plasmids pVa-C and pVa-E, with a single restriction enzyme EcoRI/XhoI double digesting pVa-C can obtain about 0.79kb capsid gene and 3kb liner pVAX1; with a single restriction enzyme BamHI or XhoI digesting pVa- E can obtain about 6kb liner pVa-E,sequencing results also show that the sequence of the constructed helper plasmids are correct.3.Quality identification of XJ-160 virus plasmid-based replicon vector system, with a single restriction enzyme NotI/AscI double digesting pVaXJ-EGFP can obtain about 0.7kb EGFP gene and 10.5kb liner pVaXJ,sequencing results also show that the sequence of the EGFP expression plasmids are correct. Co-transfected the reporter gene expression plasmid and helper plasmid into BHK-21 cells,24 hours after transfection it can expression green fluorescent protein.4.Quantity identification of XJ-160 virus plasmid-based replicon vector system, with a single restriction enzyme FseI/AscI double digesting pVaXJ-EGFP can obtain about 0.59kb GLUC gene and 10.5kb liner pVaXJ,sequencing results also show that the sequence of the GLUC expression plasmids are correct. Co-transfected the reporter gene expression plasmid and helper plasmid into BHK-21 cells,24 hours after transfection G.luc activity began to increase,30h arrived the highest value (7.8×104). In the 30-48h after transfection G.luc activity decreased significantly,48h the activity is (3.0×104). 6h-18h after transfection began to increase slowly; as G.luc rapid increase in the expression and the accumulation effect,30h form a signal peak; then decreased slowly.ConclusionWe have successfully constructed XJ-160 virus plasmid-based replicon vector system on the base of XJ-160 virus infectious cDNA clone, the replicon vector system can replicate and translate independently and express foreign protein stablely.