Establishment Of Polyhedrin Expressing Stable Cell Line That Resume Bodies Of Recombinant Bombyx Mori(silkworm) Multiple Nucleopolyhedrovirus

Over the past 20 years the baculoviruses vector expression systerm (BEVS) has beco-me one of the most widely used systerms for routine production of recombinant protein. Proteins produced in the BEVS are very similar to naturally occurring human proteins in terms of post-translational modifications (e.g,phosphorylation), biological activity, and protein stability. The culture conditions of insect cell is more convenient as compared with E. coli ,but the culture of insect cell can not ferment large-scale because of the cost。In contrast, we have a long history to breed silkworm in China, it is very simple and cheap .The cost of silkworm breeding is thenth of cell culture, while the protein expression was her ten times, so it is a good application prospects to use silkworm body express foreign proteins in efficient.All the expression of inclusion body recombinant viruses are occlusion bodies defective virus, the virus can not form inclusion bodies could only form a budding virus particles.budding virus particles can not injecte silkworm larvae throug feeding,so we must inject budded virus solution by hand to express exogenous gene.But if we have mass production,the injection has many limitations, so we save means to be adopted to achieve this, first get the cell or silkworm larvae polyhedrin gene stable expression cell lines or the silkworm strains of rescue, rescue recombinant virus infection cell lines when the cells have been expressed as polyhedrin, so can save cells assembled into the virus inclusion bodies. These are rescue recombinant virus inclusion body can be infected silkworm larvae through feeding, due to the recombinant virus genome, there is no polyhedrin gene, when silkworm larvae infected with the virus particles, they are unable to express the formation of inclusion bodies and re-polyhedrosis virus, can only be budding horizontal transmission of virus particles and infection, which continues to be high exogenous gene expression.So we can finish the test for four parts.First,we build a lepidoptera transposition helper plasmid pie2piggBac.Then, we build transposon donor vector pBac-A3ph-IE1zeo,harboring both polyhedrin gene droved by BmA3 promoter and zeocin resistant gene controlled by IE1 promoter from OpNPV vector . Second,the transposon donor vector pBac-A3ph-IE1zeo transefeced with a lepidoptera transposition helper plasmid pie2piggBac into BmN cells. A polyhedrin expressing stable cell line was constructed by screening with the 200μg/ml zeocin selection medium for one month..Third, Occlusion bodies (OB) were rescued successfully in the polyhedrin expressing BmN cells infected with recombinant BmBac-gfp budded virus without polyhedrin gene. The amount of rescued OB produced in polyhedrin expressing cells is only 8% of wild type BmNPV OB produced in normal cells.Fourth,The silkworm larvae were infected efficiently when they were feed by the rescued OB sprayed mulberry leaves. After 7 days silkworm larvae found that some of the typical symptoms of a virus infection , 10 days after foreign GFP was expressed efficiently and all the silkworm larvae to become green. Further to take the green silkworm larvae hemolymph was observed under fluorescence microscope, blood lymphocytes as a green fluorescent protein, but no OB was observed in the hemolymph of silkworm larvae. Description stable cell expression can assemble polyhedrin inclusion bodies of viruses into an effective way through the infection of silkworm feeding, but it is no polyhedra in the silkworm larvae to form, so as to maintain the foreign gene expressed sustained and efficient.