家蚕Y-Box蛋白的克隆表达以及功能分析

he Y-box proteins are a family of highly conserved nucleic acid binding proteins which exist widely in various organisms from bacteria to human.The full open reading frame (ORF) of Bombyx mori Y-box was obtained from silkworm pupa by RT-PCR and named BYB (Bombyx mori Y-box), of which the accession number was AB098537.1 After cloned into pET-28a and expressed in E.coli BL21 (DE3), the BYB was fused with His-tag and purified on chelating columns. The purified fusion protein was used to immunize a male New Zealand rabbit. Then, antiserum was harvested, and IgG was prepared by affinity chromatography using immobilized protein A.BYB in some tissues of the fifth instar larvae, such as head, Malpighian tubule, epidermis, silk glands, seminal glands, gut and fatty body, were extracted and analyzed by Western blotting. The result showed that BYB existed in larva’s seminal glands,Epidermis, and so on. And the similar results was obtained when we applied immunohistochemistry analysis to the fifth instar larva and pupa. We also compared the quantity of BYB mRNA in different tissues of the fifth instar larva and different stages of silkworm growth. These results suggested that BYB was selectively expressed in silkworm’s seminal glands, epidermis and Malpighian tubule. After subcellular localization with BYB, we found that it mainly existed in cytoplasm under normal conditions.However, after Bm5 cell had been treated with cold shock of 4℃ for 2 h, most of the BYB protein distributed in the cytoplasm and nucleus. This research laid a good foundation of further studies on BYB.