The Effect Of Optimized Recombinant Baculovirus On EGFP Expression

In recent years,with the development of research on baculovirus as an efficient expression systems and gene transfer vector,baculovirus has been widely used in the expression of heterologous proteins in mammalian cells and has shown good application prospect as an gene therapy vectors not replicating in mammalian cells.The subject attempted to add WPRE,ITRs and VSVGED to the b,aculovirus at the same time and then explored the effect of the modified baculovirus on both gene expression and coordinated regulation.It was clarified whether the CMV,CBA,EFI-? and WSSV iel promoter could initiate the expression of foreign genes in mammalian cells and avian cells,and then the promoter efficiency,action distance and target cells were explored.It was of importance to further understand the promoters of mammalian cells.In this study,the expression cassette drivered by CBA,CMV,EFI-a and iel promoters was inserted into the vector in the opposite direction of pPolH promoter.And then WPRE(wood-chuck hepatitis virus post-transcriptional regulatory element)was added into the 3'-UTR of eGFP gene,which was a post-transcriptional regulatory element boosting baculovirus-mediated gene expression in vertebrate cells.And the recombinant baculovirus was called pX-WPRE-eGFP.Then an expression cassette including VSVG or VSVGED(Truncated vesicular stomatitis virus G protein)under the control of pPolH promoter was inserted into the vector,to aim at enhancing the transfection efficiency,the recombinant baculovirus called pX-eGFP and pX-gv-eGFP.Then based on the plasmids,the construction of another transfer vector pX-I-TRs-eGFP were achieved by flanking ITRs(Adeno-associated virus invelrted terminal repeats)to the x-eGFP-WPRE expression cassette in order to prolong the expression of transgene.After getting the restriction map of the transfer vectors,recombinant transfer vectors were transformed into E.coli DH10Bac competent cells.Recombinant bacmid DNA was extracted and transfected into Sj9 insect cells and then high titer recombinant baculovirus was obtained..The different recombinant baculovirus were transfected into Sj9 insect cells,CHO-K1 cells,and chick primary cells,after 48h,the expression of eGFP in CHO-K1 and CEF cells were observed by flow cytometry and fluorescent microscopical.The results showed that the eGFP gene could be drivered and expressed by the CMV,CBA,EF1-? and WSSV iel promoters in CHO-K1 cells and chick primary cells,but the expression in different cell lines were different.In CHO-K1 cells,the effects were the CBA>CMV?EF1-?>iel;in the chick embryo primary cells,expression intensity order was CMV=CBA>iel>EF1-?.The WPRE-modfied recombinant baculovirus enhanced the expression of eGFP,and compared with the control,the rates of expression increased by 57.69%and 42.89%.Then the results showed that baculovirus with both VSVGED or VSVG and WPRE also enhanced the expression of eGFP and the expression rates increased by 81.65%and 64.90%in mammalian cells and chick embryo fiber cells respectively.The study also found that the ITRs effectively extended the eGFP expression time in the cells and the strong fluorescence could also be seen after 240h.In Sj9 cells,iel promoter had strong activity to expressed eGFP,and the expression rate of eGFP showed direct proportion with the MOI and when the MOI was 50,the expression rate of eGFP reached the highest.Moreover,the pisparity effect of VSVG on insect cells could be clearly observed and the appearence of the fusion phenominon showed direct proportion with the MOI and the infection time.When the MOI was 50,the fusion phenominon could be clearly observed after 96h,and the pisparity effect did not appear.This study will lay the theoretical and practical foundation for the further development of poultry vaccines,and provide new ideas and useful references for developing the non-replicating live vaccine against other viral diseases.