Expression And Anti-apoptotic Function Of Storage Protein 2 Gene In Silkworm, Bombvx Mori

SP2(storage protein 2)is a kind of high concentration soluble protein in silkworm hemolymph.It’s an important energy material in the growth and development of silkworm,so it was called storage protein.Several reports revealed that SP2 is not only an important energy material during the growth and development of silkworm,but also has significant anti-apoptotic effects on cell apoptosis.But there are few reports about its anti-apoptotic activity at present.Endothelial cells(EC)apoptosis is one of the important triggering factors of atherosclerosis,studying the anti-apoptotic activity of SP2 on EC has great significance for the treatments of atherosclerosis and other cardiovascular diseases.sp2 gene(GenBank accession number is DQ443358)was cloned from cDNA library of silkworm pupa constructed by our laboratory.Bioinformatics analysis results revealed that it contained a 2112 bp open reading frame(ORF),encoding a protein of 703 amino acids.The predicted protein molecular weight was 83.45 kDa and the isoelectric point was 5.7.The sp2 gene amplified by PCR was inserted into the pET-28a(+)vector,and expressed in E.coli BL21(DE3)induced by IPTG.After purified by Ni2+-affiinity chromatography,the fusion protein was used to immunize the male New Zealand rabbit to obtain polyclonal antibody.The results of ELISA showed that the antibody titer was 1:6400.Western blot analysis showed that the antibody had a good specificity.Then Western blot was used to evaluate the distribution of SP2 in different stages of silkworm and different tissues of the fifth instar larva.Our results showed that SP2 levels were higher in the pupal stage,hemolymph and fat body of fifth instar larvae,but rare in the egg and moth stages.Subcellular localization studies revealed that most of the SP2 protein was located on the cell membrane,and a little in the cytoplasm.In order to further study the anti-apoptotic activity of SP2 on vascular endothelial cell apoptosis,Bac-to-Bac system was used to construct a recombinant baculovirus—Bacmid-sp2.Bacmid-sp2 DNA was transfected into BmN cells.His-Tag fusion protein was expressed in BmN cells and identified by Western blot.Ni2+-affinity chromatography was used to purify the fusion protein,and then the purified protein was added to the cultured human umbilical vein endothelial cells(HUVEC)which was treated with staurosporine to induce apoptosis previously.MTT assay showed that SP2 could significantly enhance the viability of HUVEC.Both ELISA and flow cytometry results indicated that SP2 has anti-apoptotic effects on HUVEC apoptosis induced by staurosporine.