| The Baculovirus expression vector system(BEVS)is widely used in industrial production and academic research.Using BEVS to express eukaryotic-derived proteins has several advantages,such as mammalian-like post-translational modifications of exogenous proteins,non-pathogenicity of the virus vector to mammals,low cost,and high efficiency and high level of protein expression.As the most commonly used baculovirus,Autographa californica multiple nucleopolyhedrovirus(Ac MNPV)has 154 open reading frames on its genome,encoding not only the genes necessary for viral replication,but also some nonessential genes that are irrelevant to viral replication in cultured cells.The aim of this study is to optimize the baculovirus expression vector by knocking out of some nonessential gene segments from the baculovirus genome and overexpressing late regulatory factors for recombinant protein expression.Based on Ac MNPV-derived Bacmid,Bac III and Bac IV which were previously constructed in our lab,recombinant baculoviruses expressing exogenous protein GFP were generated in this study,and their viral proliferation ability and GFP expression in the recombinant virus infected cells were compared.The results showed that there was no significant difference in the titers of the two recombinant baculoviruses,and the total GFP expression levels were similar.However,in High Five cells which commonly used in protein industry,the GFP expression speed by the Bac IV-derived baculovirus was significantly faster than that of the baculovirus made from Bac III.Bac IV was obtained from Bac III by knocking out Ac29-33,so the results demonstrated that the deletion of Ac29-33 did not affect viral replication and the production of infectious BV,but accelerated the expression of GFP.Based on Bac IVG,which was made in our laboratory from Bac IV by replacing Ac15-16 with the GFP reporter gene,a series of non-essential gene segments in the Ac MNPV genome were respectively knocked out using the Red/ET homologous recombination system combined with the rps L-AMP reverse screening system.The results revealed that knockout of Ac68-72,Ac84-87,and Ac129-131 had no negative influence on viral proliferation and BV production.Meanwhile,respective deletion of Ac68-72 and Ac129-131 significantly increased the expression level of luciferase reporter protein.However,combined knockout of Ac68-72 and Ac129-131 on Bac IVG showed that simultaneous lack of these two non-essential gene fragments seriously reduced viral replication and BV production.It has been found in a previous study in our lab that ectopic overexpression of late expression factor 6 gene(lef-6)under the control of a moderate strength promoter from gp64 can effectively increase the expression of exogenous proteins regulated by baculovirus late promoters.In this thesis,in order to further optimize the baculovirus vector,the native lef-6gene in Bac I was modified to be controlled by the gp64-lef6 tandem promoters to overexpress the lef-6 gene in situ.By constructing recombinant baculoviruses,the expression speed and yield of exogenous protein GFP were examined in the baculovirus infected cells.The results showed that both in situ and ectopic overexpression of lef-6 improved GFP production,with the latter giving a higher yield.Then,the lef-6 overexpressing modification was made on Bac IIIG and Bac IVG,which have significantly improved protein expression ability.The data showed that only the lef-6 delivered by Bac IVG was successfully overexpressed,and the expression of the exogenous protein luciferase was not improved by both vectors.In summary,two Ac MNPV vectors,whose genomes were shortened by more than 10 kb and meanwhile maintained the virus infectivity and exogenous protein productivity,were successfully constructed in this study.The results obtained here provide a foundation for further investigation of gene functions in Ac MNPV,continued optimization of the baculovirus expression system,and applications of BEVS in protein production in basic researches and in industry. |
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