丝瓜籽核糖体失活蛋白luffin-b的基因克隆及表达  

Cloning and Expression of Luffin-b cDNA from the Seeds of Luffa cylindrica

作　　者：严明[1] 杨欣秀[1] 李臻[1] 平蓓芳[1] 张祖传[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031

出　　处：《生物化学与生物物理学报》2001年第2期205-209,共5页

基　　金：中国科学院"九五"重大项目!No .KJ.95 1 B1 6 0 6&&

摘　　要：丝瓜籽毒蛋白luffin b(LUFb)是毒性极强的植物核糖体失活蛋白 ,它的cDNA序列已由Kataoka等作了报道 ,但luffin b的基因表达至今未见报道。用RT PCR方法从未成熟的丝瓜籽中克隆了luffin b的基因 (lufB) ,并构建了表达载体 pET 2 4a(+ ) lufB ,pET 2 4a(+ ) lufB转化的大肠杆菌BL2 1(DE3)用IPTG诱导表达 ,SDS PAGE和Western印迹的结果表明重组luffin b主要存在于包含体中。用逐步透析法对包含体进行了复性 ,复性产物表现出很强的N 糖苷酶活性 ,并对兔网织红细胞裂解液的体外蛋白质合成具有较强的抑制活性 ,其IC50 约为1.2 5× 10 -10 mol/L。Luffin b with Mr. 28 kD, isolated from the seeds of Luffa cylindrica, is one of the most toxic single chain plant ribosome inactivating proteins. The cDNA sequence of luffin b was already reported by Kataoka in 1992. In this work, the luffin b gene( luf B) coding sequence was cloned from the fresh seeds of Luffa cylindrica by RT PCR, and the coding sequence of the gene was shown to be identical with that determined by Kataoka. The luf B expression plasmid was constructed by inserting the luf B cDNA fragment into vector pET24a(+), and the pET24a(+) luf B vector was expressed in E.coli by 0.5 mmol/L IPTG induction. The recombinant product, which mainly existed in inclusion bodies, was identified by SDS PAGE and Western blotting. The recombinant luffin b, which was renatured by dialysis with step by step decreasing concentration of urea, showed high activity of ribosome inactivation.

关 键 词：丝瓜籽核糖体失活蛋白 lufin-b 基因克隆 表达 免疫印迹 包含体 复性 

分 类 号：Q943.2[生物学—植物学]