The Affection Of Endonuclease MazF To The Target Protein Expression In A Baculovirus Expression System

Baculovirus expression vector system (BEVS) was Baculovirus as the vector to express foreign genes by infecting inset cells or larvae. BEVS is one of the four major eukaryotic expression systems. It has lots of advantages,such as a good safety performance, high carrying capacity of target genes, possessing post translation modification system etc. to date, it is widely applied in a variety of recombinant protein producing. However, the expression of the recombinant proteins is far less than desired level. It becomes a major consideration about how to improve the expression efficiency of recombinant proteins with baculovirus expression system.We have synthesized porcine interferon gam gene (approximately500bp) and mutated ACA to ACC (coding same amino acid) bearing in the ORF using site-directed mutagenesis techniques. The mutant gene was cloned into mazf baculovirus donor vector to obtain recombinant for Plasmid mh(b)-gaml-mazf. The mutant gam gene was transferred to Baculovirus receptor plasmid through magic method and recombinant bacmid is named as Ac-gam1-mazf. This bacmid was used to transfect sf9cell and western blot analysis showed recombinant porcine interferon gam genes was expressed in sf9cell. It is reported that mazf can recognize and cut specific sequence (ACA) in mRNA, so it can effectively reduce host protein synthesis and achieve high expression of the target proteins whose mRNAs lack ACA sites. In order to investigate the effect of mazf on the expression of target gene in BEVS, we transfected sf9cells with Ac-gaml-mazf and Ac-gam plasmids.Western blot analysis showed expression of the recombinant porcine interferon gam genes was significantly increased in sf9cells, which proved mazf can improve the exogenous expression in insect cells.To improve the expression level of foreign genes in BEVS, we plan to collect virus particles after primary transfection and then infect sf9cells repeatedly in future experiments. We aim to detect the expression of protein by Coomassie brilliant blue staining after SDS-PAGE electrophoresis and provide a new approach to achieve the improvement of the efficiency of this system for large-scale production of recombinant proteins in the future.