Construction and Application of Gene Deletion Baculovirus Vector
In order to solve the low protein production and virus passages in baculovirus expression system to accelerate the industrialization process,the v-cath,Chi A,P10 that affected protein production and FP25K,DA26 genes that related to stability of the genome were deleted by Red and Cas9 recombinant techniques in this study.At the same time,the glycosylation site that affected the formation of disulfide bonds were changed based on the structure and glycosylation prediction analysis of CSFV-E2.Finally,suspension transfection was performed to increase the titer of rescued viruses.The results showed that 95％of the mutant E2 protein was in the form of homodimer.The expression level of E2 protein was increased about 4 times by optimizing donor plasmid and deletion of viral vector gene and the passages of the recombinant baculovirus strain with stable expression of protein was extended from P7 to P20.The titer of recombinant baculovirus obtained by suspension transfection increased by about 70 times,and sufficient low viral passages were obtained,which effectively shortened the time from strain construction to protein expression and solved the problems of low protein production of baculovirus expression system and virus passages of strain seeds.Therefore,the successful construction of the gene deletion baculoviruses laid a foundation for the research and production of a subunit swine fever vaccine.
BaculovirusClassical swine fever virusgene deletionE2 protein
万方数据
维普
