Expression And Functional Analysis Of TRAPα Gene From Silkworm

TRAPαwas a glycosylated protein with the weight of 34 kD in mammal, which was under regulation of Ca²⁺and located in the endoplasmic reticulum and nuclear envelope. In human bone marrow cell lines, TRAPαof mRNA transcript was positivly regulated by granulocyte macrophage colony-stimulating factors in vivo. It is reported that there was a slightly different TRAPαexpression in mice after birth in skeletal muscle and heart cells, suggesting TRAPαproteins may play a particular role in the exercise of its functions in muscle and heart.The BmTRAPα, which encoded the protein containing TRAPαconserved domain, was identified from the cDNA library, of which the accession number was DQ311413. The length of BmTRAPαis 1389 bp. The ORF contained 840 bp, encoding a polypeptide of 279 amino acids with a predicted molecular weight of 30.8 kD. After cloned into pET-28a and transformed into E.coli BL21 (DE3), PCR and digestion with BamHâ… and Xhoâ… showed that the designed fragment was inserted correctly. Fusion protein was expressed successfully in E.coli BL21 (DE3) induced by IPTG with the final concentration of 1mM. The analysis of SDS-PAGE showed that the fusion protein His-BmTRAPαwas expressed highly in BL21 with 35 kD nearly in accord with the theory value. (His tag was weight of 3.56 kD, BmTRAPαwas weight of 30.8 kD). The BmTRAPαwas fused with His-tag and purified with chelating columns. The purified fusion protein was used to immunize a male New Zealand rabbit.The titer of the obtained antibody was over 1:12800, measured by ELISA. Then, IgG was prepared by affinity chromatography using immobilized protein A.Protein were analyzed by Western blot and ELISA, which were extracted from different stages of silkworm and some tissues of the fifth instar larvae, such as head,trachea,fatbody,Malpighian tubule,midgut,epidermis,silk glands,blood and seminal glands. The result in Western blot showed that the expression level of BmTRAPαin pupa was the highest, that there was BmTRAPαexisting in larva's fatbody,Malpighian tubule,silk glands,epidermis and seminal glands. However, none was detected in larva's blood. And we got the similar results when we carried out the semi-quantitative ELISA.With real time PCR, there is a certain difference in the level of transcription and translation by comparing the quantity of mRNA in different tissues of the fifth instar larva and different stages of silkworm. After carrying out subcellular localization about BmTRAPαin BmN cell, we found that it mainly existed in cytoplasm.