家蚕OCIAD2蛋白的表达分析及其亚细胞定位

cDNA sequence encoding ociad protein isoform 2 was found from the cDNA library constructed by our laboratory and was nominated as BmOCIAD2(GenBank accession number is DQ443385). The BmOCIAD2 gene contains 5 exons and 4 introns, and the ORF is 645 bp in length encoding a protein of 214 amino acid residues, which has one conserved domain OCIA motif . Multiple alignments and homology analysis showed that BmOCIAD2 shares more than 30% similarity with other insects’ relative proteins. The recombinant protein His-tag BmOCIAD2 was successfully expressed in E.coli BL21(DE3) and then was purified. The recombinant protein was used to produce polyclonal antibody against BmOCIAD2 for Western blot analyses. The result revealed that the protein was expressed in some developmental stages of the silkworm, such as pupa, moth and egg, also in some fully differentiated tissues of the fifth instar larvae, such as testis, ovary, Malpighian tubule and head. The result of real-time PCR revealed that BmOCIAD2 was the largest in the pupa in mRNA level, and the quantity of BmOCIAD2 mRNA in testis, Malpighian tubule, ovary, spiracle, epidermis, midgut, head, fat body, silk gland was degressive. Immunofluorescence method was employed to detect the subcellular localization of BmOCIAD2 in BmN cells. Positive signal to BmOCIAD2 was mainly found in the membrane.