家蚕BmBrat基因的克隆与鉴定

Cloning and characterization of BmBrat in silkworm, Bombyx mori

摘要:

NHL家族蛋白具有调控细胞增殖与分化的功能，在哺育动物中被广泛研究。本文克隆得到家蚕NHL蛋白家族成员BmBrat基因，通过RACE技术获得该基因cDNA全长序列为3 614 bp，其ORF为2 580 bp，编码859个氨基酸，预测其蛋白分子量为94.3 kDa，等电点为6.65。利用RT-PCR技术检测其在五龄3 d家蚕各组织表达情况，结果表明其在幼虫各组织均有表达，包括丝腺、中肠、脂肪体、马氏管等，且卵巢和头部表达量最高；胚胎时期表达谱分析显示其在胚胎发育第4天和第5天有高量表达。经原核表达、蛋白纯化及免疫小鼠后获得家蚕BmBrat多克隆抗体，且Western blotting及免疫荧光检测显示该抗体可以特异检测家蚕BmBrat蛋白；免疫荧光结果表明BmBrat蛋白定位于家蚕血细胞胞质中，为进一步研究BmBrat基因的生物学功能奠定了基础。

Abstract:

NHL proteins, which play important roles in regulation of cell proliferation and differentiation, have been extensively studied on mammals. Here, we cloned a member of NHL protein family namely BmBrat in silkworm. The full-length cDNA sequence of BmBrat was obtained by means of the rapid amplification of cDNA ends (RACE), including 3 614 bp. The ORF is 2 580 bp long, encoding a protein with 859 amino acid residues. The molecular weight is 94.3 kDa and the isoelectric point (pI) is 6.65. The BmBrat expression profile was detected by RT-PCR at L5D3 larval stage, and it was expressed in all tissues, including silk gland, midgut, fat body and malpighian tubule. However, it was highly expressed in ovary and head. The expression profile was also detected at different stage of embryo development, and reached a peak at the 4th and 5th days of the embryonic period. Anti-BmBrat polyclonal antibody was generated following prokaryotic expression, protein purification and mice immunization, which is highly specific and effective for recognizing BmBrat protein through Western blotting and immunofluorescence staining. Subcellular localization of BmBrat in hemocytes revealed that it was specifically expressed in cytoplasm. This study provides a foundation for further research of the biological function of BmBrat gene.