池蝶蚌金属硫蛋白基因的原核表达及活性分析  

Prokaryotic expression and activity analysis of metallothionein from Hyriopsis schlegelii

作　　者：王诚远[1] 胡芳琴[1] 刘国凤[1] 王军花[1] 彭扣[1] 盛军庆[1] 洪一江[1] 

机构地区：[1]南昌大学生物科学系,江西南昌330031

出　　处：《南昌大学学报（理科版）》2014年第2期161-165,共5页Journal of Nanchang University(Natural Science)

基　　金：江西省科技落地计划资助项目(KJLD12001);江西省科技星火计划资助项目(20123BBF61121);江西省自然科学基金资助项目(20122BAB204016);国家自然科学基金资助项目(31160534)

摘　　要：筛选池蝶蚌性腺转录组并运用RT-PCR方法扩增池蝶蚌金属硫蛋白(metallothionein,MT)ORF框序列。该序列有216个碱基组成,编码71个氨基酸,其中半胱氨酸含量丰富,富含金属硫蛋白典型的Cys-X(1-3)-Cys结构。多序列比对表明,池蝶蚌MT蛋白序列和其他物种MT序列有很高的同源性,与三角帆蚌(Hyriopsis cumingii)MT蛋白具有100%的同源性。将该ORF框片段导入原核表达载体pET-32a上,在IPTG诱导下成功在E.coli BL21(DE3)中融合表达。SDS-PAGE分析表明,融合蛋白大小约为27kDa,在25℃,终浓度为1mM IPTG诱导4h表达量最高,重组蛋白主要存在上清液中,纯化并获得了重组蛋白。细胞增殖实验表明,纯化的MT融合蛋白有生物活性并能有效地减轻宫颈癌Hela细胞的氧化胁迫效应。Screened the gonad transcriptome and used RT-PCR method, a metallothionein gene was amplified from Hyriopsis schlegelii. It contains 216 bp and encodes 71 aa. The deduced amino acids sequence was rich in cysteine and Cys-X（1-3）-Cys structure which was classic structure in MT genes. Multiple alignment showed high identity of MT gene from different species and shared 100% similarity with Hyriopsis cumingii. Then the fragment was subcloned into the expression vector pET-32a and was expressed in E. coli BL21（DE3） cells. The results of SDS-PAGE showed that the fusion protein was highly expressed after inducing by 1raM IPTG at 4 hours and its molecular mass was about 27kDa. The recombinant MT protein were mainly soluble and was successfully purified by affinity chromatography. The proliferation experiment showed that the purified MT fusion protein had biological activities which could effectively protect cervix cancer Hela cells from oxidative stress.

关 键 词：池蝶蚌 金属硫蛋白 原核表达 活性分析 

分 类 号：Q786[生物学—分子生物学]