家蚕丝氨酸蛋白酶抑制剂基因serpin16的表达规律及体外重组表达  

Expression Pattern,in vitro Recombination and Expression of Bombyx mori Serine Protease Inhibitor Gene serpin16

作　　者：董照明[1] 赵萍[1] 王凌燕[1] 李游山[1] 张艳[1] 夏庆友[1] 

机构地区：[1]西南大学蚕学与系统生物学研究所,农业部蚕桑功能基因组与生物技术重点开放实验室,重庆400716

出　　处：《蚕业科学》2010年第2期236-242,共7页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(No.30972147);国家重点基础研究发展计划“973”项目(No.2005CB12100);国家教育部“长江学者和创新团队发展计划”创新团队项目(No.IRT0750)

摘　　要：家蚕(Bombyxmori)的丝腺是丝蛋白合成分泌的场所,存在多种丝氨酸蛋白酶抑制剂。为探究家蚕丝蛋白的合成和保护机制,采用半定量RT-PCR的方法调查家蚕丝氨酸蛋白酶抑制剂基因serpin16在家蚕不同发育时期和5龄第5天幼虫各个组织器官中的表达特征,结果表明家蚕serpin16基因仅在4眠-5龄第6天的发育期表达,并且仅在丝腺中特异表达,其中在中部丝腺前区转录水平最高,而在中部丝腺中区和后区的转录水平较低;进一步构建pGEX-4T-1-serpin16原核表达载体,并转化至大肠杆菌(Eschevichia coli)BL21(DE3),经IPTG诱导获得融合蛋白,纯化后得到单一的目的蛋白。家蚕serpin16基因在幼虫丝腺的特异表达模式提示其可能与家蚕的吐丝过程密切相关,推测该基因在维持丝腺稳定的泌丝环境中发挥重要作用。Fibroin and sericin are synthesized in silk inhibitors reside. In order to understand mechanism RT-PCR was employed to investigate the expression gland of the silkworm （ Bombyx mori） where various serine protease of silk protein synthesis and protection in silkworm, semi-quantitative patterns of silkworm serine protease inhibitor gene serpin16 at different developmental stages and in various tissues/organs of the 5th day larvae of the 5th instar. The results showed that serpin16 was specifically expressed in silk gland from the 4th moulting to the 6th day of the 5th instar. Its transcriptional level in anterior region of the middle silk gland was the highest. The transcriptional levels in middle and posterior regions of the middle silk gland were relatively lower. Prokaryotic expression vector pGEX4T-1-serpin16 was further constructed and introduced into Escherichia coil BL21 （DE3）. Fusion protein was expressed successfully in the form of inclusion body through induction by IPTG, and further purified to obtain the single target protein. The exclusive expression pattern of silkworm serpin16 indicated that it is closely related with the process of silk spinning and may play an important role in maintaining the homeostasis for silk secretion in the silk gland.

关 键 词：家蚕 丝腺 丝氨酸蛋白酶抑制剂 半定量RT—PCR 原核表达 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]