家蚕BmAWD基因的表达和蛋白纯化  

Expression and Purification of Abnormal Wing Disc Gene from Silkworm, Bombyx mori

作　　者：赵贤能[1] 童富淡[1] 蒋彩英[1] 张耀洲[1] 

机构地区：[1]浙江理工大学生物化学研究所,浙江杭州310018

出　　处：《蚕桑通报》2009年第4期13-17,共5页Bulletin of Sericulture

基　　金：国家973计划课题(NO.2005CB121006);浙江省自然科学基金(NO.Y3080183)资助

摘　　要：家蚕BmAWD与家蚕翅膀发育密切相关,控制其翅膀发育的完整性及可用性。在对本实验室构建的家蚕蛹期cDNA文库测序中,我们筛选到一条编码AWD蛋白的cDNA序列,将其命名为BmAWD基因。通过生物信息学分析,发现该基因序列全长为689bp,ORF框为465bp,编码154个氨基酸残基,含有一个NDPK保守结构域。通过PCR扩增获得该基因ORF框并克隆到表达载体pGEX-5X-1上,得到重组表达质粒pGEX-5X-1-AWD,将该重组子转化大肠杆菌BL21,经PCR、酶切鉴定证明重组正确,在37℃下以IPTG诱导表达后收集菌体并裂解,SDS-PAGE分析发现在43kD左右处有一特异性蛋白条带,与预期值相符,但目的蛋白主要以沉淀即包涵体形式存在,25℃下诱导表达,SDS-PAGE分析表明目的蛋白主要存在于上清中。采用亲和层析法纯化融合蛋白GST-AWD,目的蛋白通过与层析柱上的GST磁珠结合而被纯化分离。BmAWD is closely related to the wing development of silkworm, it controls the the whole process of wing development. According to the large scale sequencing of cDNA library which built by our lab from silkworm pupae, a cDNA which encodes protein AWD （abnormal wing disc） was identified, so it was named as BmAWD. This cDNA sequence has a 465 bp open reading frame which encodes a polypeptide of 154 amino acids, it contains a conversed domains NDPK. The BmAWD gene was cloned and cloned into pGEX-SX-1 to constructed a recombinant plasmid pGEX-SX-1-BmAWD. E.coli BL21 was transformed with this recombinan plasmid and it confirm by PCR and restriction enzyme, induced with IPTG to express the recombinant protein under 37℃, we analysis the recombinant protein with SDS- PAGE, find that The molecular weight of this fusion protein was around 43kD, SDS-PAGE showed that the protein exist in deposition of supersonic fragmentation, so we changed to induced it under 25~C, SDS- PAGE showed that the protein exist in supernatant of supersonic fragmentation, we purify the protein with affinity chromatography. The purified fustion protein was obtained through GST affinity chromatography.

关 键 词：家蚕 BmAWD基因 核苷二磷酸激酶 基因克隆 蛋白纯化 

分 类 号：Q78[生物学—分子生物学]