凋亡素融合蛋白的可溶性表达及活性分析

Soluble Expression and Activity Analysis of Apoptin Fusion Protein

目的:构建PET-28a-SPA原核表达载体,在大肠杆菌BL21(DE3)中实现其高效可溶性表达,测定对肿瘤细胞的凋亡效果。方法:本实验在获得凋亡蛋白融合基因的基础上,成功地构建了重组表达质粒PET-28a-SPA,将阳性重组质粒转化表达受体菌BL21(DE3)感受态细胞中,经IPTG诱导表达,表达产物经聚丙烯酰胺凝胶电泳检测和Western blot检测,并采用MTT法检测其对肿瘤细胞的增殖抑制。结果:表达产物经聚丙烯酰胺凝胶电泳检测,凋亡蛋白融合基因获得高效表达,软件分析表明表达蛋白占菌体蛋白20%左右。上清表达量约为10%。上清蛋白经纯化后,Western blot结果显示,利用凋亡蛋白单克隆抗体可以很好地和所表达的蛋白带特异性结合,并且对A549肺癌细胞及Hela细胞具有一定的凋亡作用。结论:所获凋亡蛋白以高效胞质可溶形式表达,为其研制有效的肿瘤免疫治疗靶向药物提供一定的基础。

Objective：To construct the recombinant expression vector of pET- 28a- SPA and to soluble expression highly in cytoplasmic fraction in E. coli BL21 （DE3）, examine the eytotoxieity of the purified products against the cancer ceils. Methods： Recombinant expression plasmid pET- 28a- SPA was constructed according to recombinant plasmid. The correct recombinant expression plasmid was transformed into the host strain BL21 （DE3） induced by IPTG. The specific protein expressed was detected by SDS- PAGE and Western blot, the eytotoxie ability of the purified products were examined by MTT. Results： The specific protein expressed was detected by SDS - PAGE. The fusion protein was expressed at high level amounting to 20% of the total bacterial protein analyzed by computer software. The ammount in supematant is about 10%. After purified the supematant protein , Western blot showed the moneclonal antibody raised against the recombinant Apopfin in routines could react to the protein expressed specifically, and exerted eytotoxie effect on A549 and Hela cancer cells. Conclusions： The fusion protein was expressed highly in cytoplasmic fraction in BL21（DE3）, and as soluble form in E. coli BL21（DE3）. Supporting that it would be a promising candidate for tumor targeted immunotherapy.