日本七鳃鳗毒素肽rLj-RGD3突变体rLj-115基因的克隆与表达  

Cloning and Expressing of rLj-115 Gene, a Mutant Protein of rLj-RGD3 from Lamprey Lampetra japonica

作　　者：李姿其 衣龙达 米梓萌 于卓 周沫含 王继红[1] 肖蓉[1] LI Ziqi;YI Longda;MI Zimeng;YU Zhuo;ZHOU Mohan;WANG Jihong;XIAO Rong(School of Life Sciences,Liaoning Normal University,Dalian 116081,China)

机构地区：[1]辽宁师范大学生命科学学院

出　　处：《水产科学》2019年第6期868-873,共6页Fisheries Science

基　　金：国家“863”项目(2014AA093502);大连市科技攻关项目(2014E12SF067);辽宁师范大学大学生创新创业训练计划项目(cx201802055)

摘　　要：rLj-RGD3是一种含有3个RGD模体的七鳃鳗毒素蛋白,具有抗血栓和抗肿瘤功能。为研究3个RGD模体与其蛋白功能的关系,rLj-RGD3系列突变体的获得成为关键。rLj-115是第Ⅰ及第Ⅱ位RGD缺失、第Ⅲ位RGD保留的突变体,本研究针对其进行了基因克隆、蛋白表达及纯化。由于rLj-RGD3基因具有大量重复序列,突变体基因无法通过定点突变方法获得,故对该突变体基因进行人工合成及PCR扩增、NdeⅠ和HindⅢ双酶切,并构建于pET23b质粒。DNA测序结果显示,该重组质粒所构建序列正确。进一步对阳性重组子进行转化、诱导表达和亲和层析纯化,得到了纯化的rLj-115蛋白。rLj-115作用于人脐静脉内皮细胞ECV304的MTT试验结果表明,该合成基因表达的蛋白具有活性,可以用于后续研究。rLj-RGD3 as a toxin protein with 3 RGD motives from lamprey Lampetra japonica functions as anti-thrombosis and anti-tumor and it is the crucial for understanding of the relationship between functions and the 3 RGD motives to establishment of a series of mutants against rLj-RGD3.The one of these mutants,rLj-115,the mutant only with the third RGD motif,was cloned and expressed.Because there were many repeat sequences in the DNA of rLj-RGD3,the site-specific mutation was not performed,and the DNA sequence of rLj-115 was obtained by artificial synthesis and PCR amplification,and the PCR products digested with NdeI and HindⅢwere ligated into pET23b vector and identified by DNA sequencing in order to obtain recombined peptide of rLj-115.The recombinant plasmid of pET23b-115 was then transformed into Escherichia coli BL21.The purified rLj-115 peptide was obtained by IPTG induced expression and purification of affinity chromatography,and the biological activity of rLj-115 was confirmed by MTT assay of ECV304 cells,which provides the basis for the further research.

关 键 词：日本七鳃鳗 RGD毒素肽 基因克隆 诱导表达 重组蛋白 

分 类 号：Q785[生物学—分子生物学]