融合蛋白M-IL-2(^(88)Arg,^(125)Ala)在毕赤酵母中的表达及抗肿瘤活性研究  

Expression&Antitumor Activity of Fusion Protein M-IL-2(^(88)Arg,^(125)Ala) in Pichia pastoris

作　　者：李林[1] 钱冬萌[1] 邵光灿 闫志勇[1] 宋旭霞[1] 陈豪[1] 王斌[1] 

机构地区：[1]青岛大学医学院病原生物学教研室,山东青岛266071

出　　处：《微生物学杂志》2014年第2期47-53,共7页Journal of Microbiology

基　　金：山东省科技攻关项目(2007GG3WZ05009);山东省优秀中青年科学家科研奖励基金(BS2011SW005)

摘　　要：构建融合基因蜂毒肽(melittin)与人变构白介素2(M-IL-2(88Arg,125Ala))毕赤酵母分泌型表达载体pPICZαA/M-IL-2(88Arg,125Ala),转化毕赤酵母菌株KM71H,甲醇诱导筛选出的多拷贝阳性菌株表达融合蛋白,纯化融合蛋白,并进行初步的抗肿瘤活性研究。经测序及PCR鉴定,M-IL-2(88Arg,125Ala)正确插入pPICZαA中,电转化后,重组载体通过同源重组整合到酵母基因组中,SDS-PAGE检测在26 ku左右有明显目的条带,与理论相符。经Western blot鉴定具有较高抗原性及特异性。BCA法测定甲醇诱导96 h融合蛋白表达量达315.2 mg/L。CCK-8法检测融合蛋白对人卵巢癌细胞SKOV3细胞及Hela细胞生长抑制作用,表明纯化的融合蛋白在体外能抑制人卵巢癌细胞SKOV3细胞及Hela细胞的生长增殖。该研究为融合蛋白M-IL-2(88Arg,125Ala)大规模制备和动物实验以及临床前期研究奠定了基础。Pichia pastoris expression vector of excretive type pPICZα A /M-IL-2（88Arg,125Ala） was constructed by fusion gene melittin and human metamer interleukin 2（M-IL-2（88Arg,125Ala）） in P.pastoris,and transformed P.pastoris strain KM71H,methanol-induced and screened out a multicopy positive expression fusion protein,purified the fusion protein,and carried out initial study on its antitumor activity.It was identified through sequencing and PCR that M-IL-2（88Arg,125Ala） was correctly inserted into pPICZαA and after electro-transformation,the recombinant vector was integrated into the genome of the yeast through homologous recombination,and it was tested by SDS-PAGE that there was an obvious band of 26 ku that accorded with theory.It had fairly high antigenicity and specificity through Western blot.The methanol-induced for 96 h protein expression was as high as 315.2 mg /L through BCA testing method.The fusion protein was tested by CCK-8 method for its inhibition against human oophocarcinoma cell SKOV3 and HeLa cell,suggested that the purified fusion protein could in vitro inhibit the growth and proliferation of oophocarcinoma cell SKOV3 and HeLa cell.The study had laid a foundation for large-scale preparation of fusion protein of M-IL-2（88Arg,125Ala） and animal experiment as well as early stage of clinical research.

关 键 词：M-IL-2(88Arg 125Ala) 毕赤酵母 表达 纯化 抗肿瘤活性 

分 类 号：Q789[生物学—分子生物学]