Transformation And Application Of Novel Baculovirus Expression Vector

Baculovirus is a kind of double-stranded DNA virus with encapsulated membrane.With the development of its genome,it has been modified and developed into another expression vector besides yeast,escherichia coli and mammalian cell expression system.Currently,the commercial baculovirus expression vector USES polyhedrons or P10 promoters for high expression of exogenous genes,and can produce a large number of exogenous proteins by suspending adherent cells through infection.Due to the lack of polyhedrons,it can only infect the silkworm bioreactor by injection,which limits its application.The baculovirus expression vector of polyhedron gene supplementation was constructed,and the expression of polyhedron protein was driven by the UAS/GAL4 system to achieve the controlled expression of polyhedron gene in the silkworm bioreactor,so as to solve the problem that the recombinant virus could not be infected by mouth.At the same time,in order to improve the expression of exogenous proteins in baculovirus genome,it is not necessary to knock out some genes that affect the expression of exogenous proteins.Bombyx mori is one of the most widely used economic insects.The recombinant virus obtained by gene recombination can be infected into silkworm cells or silkworm individuals to achieve stable expression of exogenous genes,and the silkworm bioreactor with Chinese characteristics is currently the most potential insect baculovirus expression system for producing biological protein products.Therefore,the construction of a simple and efficient protein expression system will lay a foundation for the mass expression of exogenous proteins.In this study,a uas-polyhedrin /GAL4 expression system was constructed.EGFP and the capsid proteins of FMD virus were used as exogenous proteins to verify the expression ability of the system.In addition,the expression and stability of exogenous proteins were improved by knockout of non-essential genes of baculovirus expression vectors.The research results obtained are as follows:1.Verification of UAS-polyhedrin / GAL4 expression of polyhedrin in silkworm cellsUsing the wild-type BmNPV genome as a template,the gene sequence of the polyhedron was obtained by PCR.It was cloned into a plasmid containing the UAS sequence,and then the UAS-Polyhedrin-SV40(UPS)expression cassette was cloned into the p Fast Bac Dual vector,and the expression cassette was cloned into commercial silkworm baculovirus for expression by the Bac-to-Bac system In the vector,the recombinant baculovirus Bm-UAS-Polyhedrin containing UAS-Polyhredrin was obtained.After co-transfection of recombinant virus and recombinant baculovirus containing GAL4 in silkworm cells for 72 h,a large number of polyhedrons were observed.Using green fluorescent protein as the exogenous protein,the harvested polyhedron was fed to the 5th instar silkworm.After 4 days,the silkworm was found to have symptoms of infection.A large amount of green fluorescent protein could be detected in the hemolymph,but no polyhedron was found.2.Verification of UAS-polyhedrin / GAL4 expression of polyhedrin in silkworm individualsRecombinant baculovirus containing UAS-polyhredrin cassette was injected and infected into GAL4 transgenic silkworm individuals and ordinary silkworms,and blood was collected 72 hours later to observe that no polyhedrons were formed in ordinary silkworms.Compared with the amount of polyhedra formed in silkworm cells,only a few polyhedra were formed in GAL4 transgenic silkworms.3.Establishment of GAL4 transgenic cell line and expression of foreign proteinsConstruction of pBac-[39k-GAL4-sv40]-[IE2-DsRed-sv40],pBac-[IE2-GAL4-sv40]-[IE2-DsRed-sv40],pBac-[39k-GAL4-sv40]-[IE2-DsRed-P2A-Neo-sv40] t ransgene expression vector.After co-transformation of the above vector with p S L-UAS-EGFP,green fluorescence was observed,indicating that the constructed GAL4 transgene expression vector can interact with UAS expression and normal ly express protein.Subsequently,through G418 resistance screening,GAL4 trans genic cell line was obtained.The recombinant virus Bm-UPS-EGFP and the rec ombinant virus Bm-UPS-Mya98 containing the virus-like particle expression cass ette of the FMD Myanmar 98 virus strain were added to the GAL4 cell line.T he protein expression level is acceptable,and all can form polyhedron,indicatin g that the transgenic cell line has been successfully established.4.Knockout of unnecessary genes in the silkworm baculovirus expression vectorUsing Red / ET recombination technology,non-essential genes Bm104 and Bm130 on the silkworm baculovirus expression vector were knocked out,and mutation vectors have been obtained.Using the mutant vector and the exogenous protein EGFP,the replication and expression ability of the mutant vector was detected.