木瓜凝乳蛋白酶基因的克隆及在原核细胞中的表达  

Cloning of Chymopapain Gene and Its Expression in E.coli

作　　者：蔡勤安[1] 于志晶[1] 尚丽霞[1] 许诺[2] 曾军[1] 马瑞[1] 刘艳芝[1] 

机构地区：[1]吉林省农业科学院农业生物技术研究所,长春130033 [2]吉林大学生命科学学院,长春130012

出　　处：《吉林农业科学》2015年第3期23-25,79,共4页Journal of　Jilin Agricultural Sciences

摘　　要：以青番木瓜总RNA为模板,采用RT-PCR技术扩增出木瓜凝乳蛋白酶基因CHY(chymopapain),构建带有His-tag的原核表达载体p EASY-E1-CHY。将该重组载体转化到大肠杆菌BL21(DE3)菌株中,重组菌株用IPTG诱导表达,在诱导6 h,IPTG浓度为0.05 mmol/L时重组蛋白表达量最高。SDS-PAGE凝胶电泳和West-ern-Blot杂交分析结果表明蛋白大小为45 k Da。用脱脂奶粉进行凝乳分析,证明此木瓜凝乳蛋白酶具有凝乳活性,此结果为今后利用生物技术进行木瓜凝乳蛋白酶工程化生产奠定了基础。Chymopapain gene （CHY） was cloned from papaya by RT-PCR, and constructed into pEASY-E 1 to generated recombinant plasmid pEASY-E1-CHY. The plasmid was transformed into E.eoli. The expression was in- duced by IPTG and the induction time was optimized. The condition of suitable expression was 6h and 0.05 mmlo/L IPTG. The analysis of SDS-PAGE and Western-Blot indicated that the molecular weight of protein was 45kDa. The ehymopapain was expressed successfully in BL21 （DE3）and the enzyme activity was identified by curd experiment. The result will be helpful to the chymopapain engineering.

关 键 词：木瓜凝乳蛋白酶 基因克隆 原核表达 活性检测 

分 类 号：Q946[生物学—植物学]