Construction Of Efficient Expression Vector Of Baculovirus And Its Expression Of EGFP In Chicken Embryonic Primary Cells

The Bac-to-Bac Baculovirus Expression Vector System is a class of eukaryotic expression system. Recently it has been used extensively as a transfer vector for mediating the expression of recombinant proteins in mammalian cells in biology, medicine and other fields. The baculovirus does not initiate replication cycle in mammalian and guarantees very high biosafety. What`s more, the tropism to different kinds of cells and the transduction efficiency of baculovirus even can be obversly improved by introduction of cell-specific promoter, pseudotyped baculoviruses, adding different functional regulatory elements and so on.Within this study, two recombinant baculoviruses were constructed with CMV promotor, VSVGED-pseudotyped, WPRE and ITRs on the base of Bac-to-Bac baculovirus expression vector system, and the protein expression level in chick embryo primary cells respectively mediated by the above two baculovirus was studied and analyzed.(1) The target DNA sequence named“PH”,“gp64SP”,“TM/CTD”,“CMV”,“WPRE”and“pA”were amplified separately by specific-designed PCR primer, and then inserted into the backbone plasmid pFastBac[PH] by the combination of SOE-PCR and the traditional digestion-connection methord to construct the recombinant transfer vector“pLM”. (2) The target DNA sequence ITRs were also ampilied by specific-designed PCR primer, and then inserted into pLM by digestion-connection methord to construct the recombinant transfer vector“pLM-ITRs”. (3) Recombinant trasfer plasmids pLM-eGFP and pLM-ITRs-eGFP were constructed by inserting reporter gene eGFP into the transfer vector constructed above and then respectively transformed into the E.coli DH10Bac competent cells in order to generate the recombinant bacmids Bac-LM-eGFP and Bac-LM-ITRs-eGFP with the help of BEVS. (4) Then the two recombinant bacmids above were transfected into Sf9 insect cells respectively to generate the recombinant baculovirus P1 stocks of BV-LM-eGFP and BV-LM-ITRs-eGFP. (5) Continue to amplify P1 stock up to P3 and then calculate the virus titer by Plaque Assay(BV-LM-eGFP: 1.1×108 pfu/mL; BV-LM-ITRs-eGFP: 1.2×108 pfu/mL). (6) At last, the chick embryo primery cells were infected separately by BV-LM-eGFP, BV-LM-ITRs-eGFP and the control virus BV-FB-eCMV-eGFP , and then the expression level of eGFP was observed and analyzed.The eGFP expression results showed that (1) VSVGED-pseudotyped can improve the transduction efficiency and (2) the WPRE also can increase the reporter gene eGFP expression level mediated by baculovirus in chick embryo primary cells. Thus,a firm theoretical and experimental foundation is laid for the further systematic and quantified research on the reporter gene expression level influenced by VSVGED- pseudotyped virus and by the application of WPRE.