家蚕B类清道夫受体BmSCRB8基因的克隆及表达

Cloning and expression of scavenger receptor class B BmSCRB8 in silkworm Bombyx mori

摘要:

B类清道夫受体在动脉粥样硬化及其他心血管疾病的形成或抑制，机体免疫防御，凋亡细胞清除等生理过程中起着重要的作用。克隆了家蚕B型清道夫受体家族的一个成员BmSCRB8基因，通过RACE技术获得BmSCRB8的cDNA全长为2 668 bp，其ORF为1 704 bp，编码567个氨基酸，通过在线预测其蛋白分子量为63.87 kDa，等电点为6.06。采用RT-PCR方法得到了BmSCRB8的时空表达谱，结果表明BmSCRB8在家蚕各组织以及血液各时期均有表达，且在脂肪体中表达量最高，变态发育时期表达量较高。原核表达获得BmSCRB8重组蛋白，并经由蛋白纯化、免疫小鼠后制备得到家蚕BmSCRB8多克隆抗体。同时构建了BmSCRB8真核表达载体并转染家蚕胚胎细胞系。免疫荧光及过表达结果显示BmSCRB8主要定位于细胞膜上，Western blotting结果显示免疫小鼠后所得到的抗血清可特异性识别BmSCRB8蛋白。

Abstract:

Scavenger receptor class B is involved in various indispensable physiological processes, like the formation and inhibition of atherosclerosis or other cardiovascular diseases, innate immune defense and the removal of apoptotic cells. Here, we cloned BmSCRB8, a member of scavenger receptor class B in silkworm. We obtained the full-length cDNA sequence of BmSCRB8 by rapid amplification of cDNA ends (RACE), including 2 668 bp. The ORF of BmSCRB8 is 1 704 bp, encoding 567 amino acids. Online software prediction indicated that the molecular weight of BmSCRB8 is 63.87 kDa and the isoelectric point (pI) is 6.06. The space-time expression profile of BmSCRB8 was detected by reverse transcription PCR (RT-PCR), which implicated that BmSCRB8 is extensively expressed in each tissue and at each stage of blood. In addition, BmSCRB8 is highest expressed in fat body of silkworm, and is highly expressed in metamorphosis periods. Anti-BmSCRB8 polyclonal antibody was generated through prokaryotic expression, protein purification and mice immunization. Simultaneously, we constructed BmSCRB8 eukaryotic vector and then transfected embryonic cell line of silkworm. Immunofluorescence and overexpression showed that BmSCRB8 expressed specifically in membrane. Western blotting demonstrated that BmSCRB8 protein can be specifically recognized by anti-serum generated after mice immunization.