Construction And Identification Of Helper Plasmids For Ecombination Of Newcastle Disease Virus Italien Strain

Taking oncolytic virus (ov) as a therapeutic tool to kill cancer is a hotspot in the cancerbiotherapy field nowadays, there have a few natural and artificial oncolytic viruses beenapplied into cancer therapies research,and after gene recombinant of the natural oncolyticvirus to obtain securer recombinant oncolytic virus is a new strategy for oncolytic virusresearch. Newcastle disease virus，(NDV) is a kind of natural oncolytic virus，which iswith the character of selective replication in the human cancer cells but withoutpathogenicity in the normal cells. With the technique of reverse genetics, a recombinantvirus which is carrying exogenous gene could be reconstructed. This research tookstrong pathogenicity NDV strain and oncolytic strain Italien as the subjects to elucidatethe oncolytic characteristics in vitro,meanwhile constructed nucleocapsid protein(NP),phosphoprotein(P), and large protein(L) ukaryotic expression plasmid,and the virusminiature genome plasmid based of phage T7promoter as the helper plasmids for futurevirus recombinant. Through this research we have primary understanding of the NDVItalien oncolytic character and did solid spade work for future recombinant work.This research contains three parts:Part1: The cell viability caused by NDV ItalienObjective: The cell viability was measured in tumor cells infected with NDV Italien.Methods: NDV Italien was inoculated into the allantoic cavity of specific pathogenfree embryo. The allantoic fluid was collected at3day-post inoculation. Virus was purifiedby high-speed centrifugation followed with ultra-speed centrifugation. The virus titer wasvalued by method of median tissue culture infective dose (TCID50). Cell fusion wasobserved in HeLa cells by Hematoxylin-eosin(HE) staining infected with NDV Italien. Thecell viability was measured with MTT assay in HeLa cells infected with different titers ofNDV Italien.Results: The titer of purified virus suspension was1010TCID50/ml. NDV induced cytopathic effect characterized by syncytia formation when observed through HE staining.The MTT experiment indicated that the cell viability was significantly declined afterinfection with NDV Italien.Part2: the construction of helper plasmids for NDV Italien strainObjective：To construct the NP,P and L ukaryotic expression plasmid of NDV ItaliensrainMethods: To culture virus by infection of chicken embryo via chorioallantoic cavity,and purify and condense viruses by super centrifuge. Extract the virus genome RNA byusing RNA extraction kit, using high fidelity RT-PCT to obtain virus, and NP，P and L.Meanwhile using high fidelity PCR to obtain internal ribosome entry sites(IRES) carryingT7promoter element and EGFP coding sequence fragment, and link them into the T vector,then obtained the plasmid T7-test. Later the NP,P and L gene replace the EGFP of theT7-test by digestion to obtain NP，P and L eukaryotic expression plasmids, and finallytransfect there eukaryotic expression plasmids into BSR-T7/5cells which could expressstable T7polymerase,and test the protein expression by indirect immunoflorescence.Results: After chicken embryo chorioallantoic cavity culture and virus purification weobtained high concentration virus suspensions; We extracted the virus genome RNA byusing RNA extraction kit, obtained the plasmid T7-test carrying T7, internal ribosome entrysites(IRES), and EGFP, transected this plasmid into BSR-T7/5cells the expression ofEGFP could be detected,which indicated that the plasmid could express downstream genewith high output by using T7RNA polymerase. After digestion we obtained the NP，Pand L eukaryotic expression plasmids, and named them T7-NP，T7-P and T7-L. Thetransfection of these plasmids into BSR-T7/5cells, the virus proteins expressed stably.Part3: The construction of miniature genome plasmids and the functionverification of the helper plasmids.Objective: To reconstruct the miniature genome plasmids carrying report gene and thefunction verification of the helper plasmids.Methods: To synthesis the HDVrz sequence of the HDV, using the RT-PCT to amplifythe3’ and5’ non-coding sequence, then linking T7promoter,the virus non-codingsequence and the report gene one by one using overlap PCR, and insert this product intothe plasmid containing HDVrz sequence after digestion to obtain the virus miniature genome plasmid. To infect the BSR-T7/5cells which is already transfected with theminiature genome plasmid with NDV,to investigate the expression of the report gene toverify the function of the miniature genome. Finally,to verify the function of the helperplasmids system by co-transfection of miniature genome with the helper plasmids into theBSR-T7/5through watching the report gene expression.Results: We obtained the recombinant plasmid pUC-HDVrz after insert the HDVrzafter artificial synthesis, meanwhile added restriction enzyme sites in its upstream,thewhole3’ and5’ non-coding sequence of the virus genome was amplified by RT-PCR,and linked the firefly luciferase gene and T7promoter sequence in the up-stream by overlapPCR. We linked the miniature genome fragment into the pUC-HDVrz after digestion andobtained the virus miniature genome plasmid and named it MG-L. After transfection of thisplasmid into BSR-T7/5cells and infect the cells with NDV the report gene could bedetected,which validated the miniature genome could transcript the report gene by usingthe virus proteins. Then36hours after the co-transfection of MG-L and helper plasmidsinto the BSR-T7/5cells the firefly luciferase could be detected,which indicated theproducts of the helper plasmids could form functional RNP complex and transcript thereport gene of the miniature genome.Conclusions: This research validated the in vitro character of killing cancercells,meanwhile successfully constructed the helper plasmids which could be used foroncolytic virus NDV recombinant,the helper plasmids using the high efficiency phage T7promoter liked with IRES for the first time which could specifically express the virusprotein the eukaryotic cells.At the some time successfully constructed the virus miniaturegenome plasmid carrying report gene,and using the plasmid verified the whole functionof the helper plasmids, which is a solid spade work for future recombinant virus revivework.