LMP1 and green fluorescent protein fusion gene Preparation of lentivirus.docVIP

 PAGE \* MERGEFORMAT 13 LMP1 and green fluorescent protein fusion gene Preparation of lentivirus Authors: Li Xianmao, ZHAO Tong, ZHU Mei-Gang, ZHANG San-quan, ZHANG Jin-Hua [Keywords:] Polymerase chain reaction Production of lentivirus of LMP1 and GFP recombinant gene [Abstract] AIM: To produce lentivirus of latentmembrane protein 1 (LMP1) and green fluorescent protein (GFP) recombinant gene. METHODS: The fragment including all the exons of LMP1 was amplified by RTPCR and was recombined to the downstream of CMV promoter in the lentivirus vector FUGW. The three plasmids expressing lentivirus [packaging plasmid pCMV. △ 8.9, envelope plasmid VSVG and target plasmid FUGWLMP1] were packaged into virus packaging cell line 293FT using lipofectamine method. The virusproducing cell supernatant was harvested and concentrated. RESULTS: It was proved that the sequence of the RTPCR product was totally consistent with the data of GenBank by DNA sequencing analysis. The LMP1 cDNA fragment was cloned in the vector FUGW in the right direction and the open reading fragment of LMP1 was maintained. The three plasmids were effectively transferred into 293FT. Much green fluorescence was observed by fluorescence microscopy. CONCLUSION: The lentivirus of LMP1 and GFP recombinant gene are successfully produced. [Keywords] latent membrane protein 1; polymerase chain reaction; lentivirus [Abstract] Objective: To prepare the EB virus LMP1 and green fluorescent protein (GFP) fusion gene lentivirus. Methods: RT PCR method to obtain the whole LMP1 exon fragments, so that with a green fluorescent protein cloned into the lentiviral expression plasmid FUGW in . by restriction enzyme digestion, PCR amplification and sequencing recombinant vector. liposome-mediated lentiviral packaging systems will be the packaging of structural gene pCMV. △ 8.9, envelope gene VSVG, the target gene FUGWLMP1 into virus packaging cells 293FT , fluorescence microscopy detection of gene expression and packagi