Construction Of The Reverse Genetic Recombinant Vector Of Vesicular Stomatitis Virus

Vesicular stomatitis virus (VSV), the prototype of Rhabovirus, is an enveloped,nagative strand RNA virus. The whole genosome of the Indiana serotyes is 11161bp.Many animals can be infected by VSV, but naturally occurring human infectious arerare. Most VSV infectious are asymptomatic in humans, although mild flu-likesymptoms have been reported in some individual. With the developing of reversegenetic methods, VSV is becoming a useful therapeutics. As an oncolytic virus, VSVhas several advantages over other virus. Firstly, oncolytic VSV has no transformingabilities. Secondly, oncolytic VSV is not gene attenuated and its oncolytic abilities isvery effective. Thirdly, VSV can be used as an anti-cancer agent in all types of tumors.VSV has also been developed as a vaccine vector. All the vaccines are based on thelive, attenuated VSV. The VSV vaccine vector also has several advantages, such as thesimplified structure, the large capacities of accommodation of the exogenous gene (theinserted fragment can reached 4.5 kb), the mucosal administration manner, etc. In thisregard, VSV therapeutics are expected to find a wide application in the areas ofbiological agents at cellular levels for the treatment of human disease. In the presentpaper, the whole cDNA, L protein gene, N protein gene and P protein gene of the VSVwere cloned. The reverse genetic system of VSV was established by construction ofthe vector including the whole cDNA of VSV and the expression vector of L protein,N protein and P protein.Cloning and sequencing of the whole cDNA, L protein gene, N protein gene andP protein gene of the VSV The genosome RNA of VSV was extracted by Trizolreagent. The fragments of whole cDNA, L protein gene, N protein gene and P proteingene of the VSV was obtained by RT-PCR, the result was as expected. The fragmentswere ligated with pMD18-T vector and transformed into competent cells of TOP10.Plasmids were isolated from positive clones by alkaline lysis and identified byrestrictively digests and PCR amplification. The target plasmids were sequenced. Theresult was agreed with the sequence on the GENEBANK. So the whole cDNA, Lprotein gene, N protein gene and P protein gene of the VSV were cloned successfully.Construction of the whole cDNA reverse genetic vector of VSV PVAX1 plasmidwas engineered by adding two autonomic cut ribozyme of hammerhead ribozyme andHepatitis delta ribozyme. The obtained plasmid was named PHH. The obtainedvarious fragments of VSV cDNA were ligated together and then ligated with PHH toget the reverse genetic vector of VSV, named PHH-VSV.Construction of the expression vectors of L, N and P protein and testing of theirtranscripion in the BHK-21 cells The L, N and P protein gene was ligated withPVAX1 respectively, getting PVAX1-L,PVAX1-N and PVAX1-P expression vectors.The vectors were transfected to the BHK-21 cells by the means of liposome. The resultof transcription was detected by RT-PCR.Initially exploration of the reverse recombinant of VSV The interferonα2agene and green fluorescence protein was inserted into the PHH-VSV plasmid,gettingthe PHH-VSV-IFN-GFP plasmid. The PHH-VSV-IFN-GFP,PVAX1-L,PVAX1-NPVAX1-P was transfected to BHK-21 cells with the ratio of 16:4:2:1. The transcripionof PHH-VSV-IFN-GFP was detected by RT-PCR, the result was as expected.However, no recombinant virus was detected by electron microscope and fluorescencemicroscope. Further investigations are carrying out.