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-24a(+)-lufB，pET-24a(+)-lufB转化的大肠杆菌BL21(DE3)用IPTG诱导表达，SDS-PAGE和westem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 25×10-10 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 Katao ka in 1992. In this work, the luffin-b gene(lufB) 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 lufB expression plasmid was constructed by inserting the lufB cDNA fragment into vector pET24a( + ), and the pET24a( + )-lufB 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 blot ting. The recombinant luffin-b, which was renatured by dialysis with step by step decreasing concentration of urea, showed high activity of ribosome inactivation.
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