Optimization Of The Baculovirus Expression Vector System By Knocking Out Nonessential Viral Genes And Inhibiting Cell Apotosis

The baculovirus-insect cell expression system has the ability to express foreign genes efficiently in a eukaryotic expression environment and the post-translational modification processing of foreign proteins.And the baculoviruses are non-pathogenic to mammals.These characteristics make the expression system has been widely used in many fields.Over the years,in order to increase the efficiency of virus recombination and the expression yield of proteins,researchers have modified the expression system continuously.Based on previous researches,this study utilized molecular biology techniques and adopted two strategies to optimize the baculovirus expression vector.First,using the Red/ET homologous recombination system combined with rps L-AMP reverse screening system to knock out a series of non-essential genes in the AcMNPV genome.Minimize the baculovirus genome size and endow it the ability to insert large foreign fragments.The study found that after knocked out Ac11-13,Ac15-16,Ac18-23,Ac29-33,Ac63-64,Ac68-72 and Ac84-87 genes in baculovirus expression vectors,the expression levels of foreign proteins increased significantly.This study also indentified that the Ac12,Ac33,and Ac118 genes are non-essential genes because they are do not affect the virus multiplication in cell culture in vitro when they are deleted.Whether expressing intracellular proteins or secreted proteins,the deletion of the Ac84-87 gene could increase the expression of foreign proteins.In addition,the knock out of the six oral infectious factors(PIFs),such as Ac22,Ac68,Ac96,Ac115,Ac119,and Ac148 genes also improved the biosafety of baculovirus vectors on the basis of they were not affect the viral multiplication.The above results lay a foundation for further increasing the capacity of the expression vector to accommodate the exogenous fragments,increasing the expression and activity of foreign proteins,and studying the function of AcMNPV genes.Second,the recombinant baculovirus carrying the shRNA expression cassette which can against the host cell caspase-1 was constructed to prolong the expression time of the exogenous proteins through the anti-apoptosis strategy of the host cell,thus increasing the expression level of the foreign proteins.Previous studies in the laboratory showed that using recombinant baculovirus carrying Sf-caspase-1 shRNA gene and related expression elements can express interfering RNA in Sf9 cells,thereby inhibiting baculovirus-induced cell apoptosis.When using this system to express luciferase,the anti-apoptotic strategy did not significantly increase the protein expression,but the luciferase activity increased significantly.In this study,recombinant baculoviruses expressing the foreign proteins GFP and DsRed were constructed.The expression of the foreign proteins GFP and DsRed and their fluorescence signals were detected.Moreover,the detection results showed that the anti-apoptotic strategy may promote the folding of the proteins correctly,rather than increase the translation efficiency of exogenous proteins at the later stage of baculovirus infection.On this basis,by the alignment of the caspase-1 gene in Sf9 cells and High Five cells,we designed and constructed an AcMNPV shuttle vector carrying an expression cassette against caspase-1 in both cell lines,leading to the inhibition of apoptosis both in Sf9 cells and High Five cells.The experiment results showed that the baculoviruses prepared with the vector carrying the shRNA expression cassette were infected with Sf9 cells or High five cells,and the expression levels of the foreign proteins GFP,Fluciferase,OD-Fc,GP5-Fc or IHNV-N were all increased significantly.Studies have shown that this baculovirus vector can be used to produce various recombinant proteins in insect cells,in particular for the expression of active proteins for functional and structural studies or therapeutic applications.