Study On Expression Of WPRE And ITRs Vector Driven By EF1_α Initiated By Pseudotyped Baculovirus

Baculoviruses expession system develpoed by Invitrogen company is the most popular applied gene expression system for its much more convenience.With the possible exception of the advangtange with the Baculoviruses in the field of gene expression, gene therapy and tissue engineering,the short expression period,low expression efficiency and the low transduction efficiency are the key limits of its application in practical production .Two improved recombinant Baculoviruses transfer vector named pWK and pWK-ITR were constructed with the pFastBac1.The G glycoprotein gene of the vesicular stomatitisvirus(VSV-G) routinely used to enhance the target range was promted by pPolH promoter which can enhance the transduction efficiency of the Baculoviruses.The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is a powerful viral enhancer element that is thought to improve virus gene expression by modification of RNA polyadenylation, RNA export and/or RNA translation. WPRE was inserted into the the 3’UTR of the target gene and the vector constructed above was named pWK. Adeno-associated virus (AAV) inverted terminal repeats (ITRs) flanking EF1αcassette was constructed by inserting L-ITR and R-ITR into pWK by enzyme digestion respectivly which can make a long-lasting expression period. The vector construcred above was named pWK-ITR. The control vector pWK(-) was constructed only with the EF1αexpression cassette.The recombinant transfer vector pWK-EGFP ,pWK-ITR-EGFP and pWK(-)-EGFP was constructed by subcloning the EGFP gene into pWK and pWK-ITR. The recombinant plasmid was transformed into E.coli DH10Bac competent cells. And then the extracted recombinant bacmid DNA transfected Sf9 insect cells in order to acquire recombinant baculovirus carrying EF1α-EGFP expression cassette and the recombinnant virus was named BV-WK1,BV-WK2,BV-WK3.The titer of P3 baculovirus stock determined by a plaque assay was 1.23×108 pfu/mL,1.04×108 pfu/mL,1.32×108pfu/mL respectivly. The expression of EGFP could be detected 12h after OL cells were infected by BV-WK1, BV-WK2 with 40 multiplicities of infection. The expression of EGFP reached the highest level between 48h and 72h after infection. The EGFP expressed by the control virus BV-WK3 is about 72 hours less than BV-WK2. The transduction process of the control virus BV-WK3 was about 24 hours more than BV-WK1, BV-WK2 .The percent of the positive cells expressing EGFP was separately 57.3%,69.1%, 32.6%.So our method suggests that recombinant baculoviruses could efficiently deliver its genome DNA into OL cells and the reporter gene could be successfully expressed by the function of the mammalian-cell-active promoter EF1α. WPRE,ITRs,VSG-GED are the promident factors for the gene delivery and expression.