Optimization Of The Baculovirus Expression Vector By Anti-apoptosis

Baculovirus expression vectors can efficiently express heterologous proteins, which use baculovirus very late gene promoter such as polyhedrin gene promoter or p10 gene promoter. And baculovirus-insect cell expression system can be modified at post-expression, so it is widely used to express biologically active proteins. Nevertheless, in practice, a lot of proteins yield is still low. Therefore, it is meaningful to build a more efficient baculovirus expression vector, to provide more target protein for the structure and function study.In this study, chitinase gene(chiA) and cathepsin genes(v-cath) of AcMNPV Bacmid were replaced by rpsL-AMP fragment to get a new knockout bacmid. This laid an important foundation for subsequent experiments.Then inverted repeat sequence inserted into pBac5 to express Sf-caspase-1 full-length double-stranded RNA(dsRNA) and then was co-transfected with AcMNPV Bacmid into Spodoptera frugiperda 9(Sf9) cells to produce recombinant baculovirus AcMNPV-dsCasp. AcMNPV-dsCasp can express Sf-caspase-1 full-length dsRNA and inhibit the apoptosis of Sf9 cell. The result showed that Sf9 cell infected by AcMNPV-dsCasp decreased Sf-caspase-1 mRNA content to successfully inhibit apoptosis.In addition, we designed Sf-caspase-1siRNA to interfere Sf-caspase-1 expression. Three Sf-caspase-1 different inverted repeat sequence, 124, 422 and 783, were insected into the plasmid pTriExGFP in order to express Sf-caspase-1 shRNA. Then three plamids co-transfected with AcMNPV Bacmid to produce recombinant baculovirus AcMNPV-124/422/783, respectively. AcMNPV-GFP-ScaI-124/422/783 can express Sf-caspase-1 shRNA and inhibit the apoptosis of Sf9. PI staining results showed that fragment 124 is the most significant inhibiting the apoptosis of Sf9 cell in the three Sf-caspase-1 shRNA.In order to construct baculovirus expression vector which expressed Sf-caspase-1 shRNA, we gained AcMNPV Bacmid-124/422/783 which Sf-caspase-1 different inverted repeat sequence was inserted into AcMNPV Bacmid backbone. AcMNPV Bacmid-124/422/783 and pTriEx-Fluc co-transfected to generate recombinant virus, detected Sf9 cell apoptosis with PI staining.In this study, expressing Sf-caspase-1 full-length dsRNA and shRNA interfered Sf-caspase-1 gene, and inhibited Sf9 apoptosis. Besides, short inverted repeat sequence of expressing Sf-caspase-1 shRNA integrated into AcMNPV bacmid skeleton. AcMNPV Bacmid-124/422/783 can express exogenous proteins and is capable of inhibiting apoptosis protein Sf9 and extending time of expressing proteins. So it provides an important foundation to enhance the foreign protein.