超抗原SEA与抗黑色素瘤ScFv融合基因的构建及表达  

The Construction and Expression of Superantigen SEA and Antimelanoma ScFv Fusion Gene

作　　者：孙静[1] 吕安国[1] 吴文芳[1] 白向阳[1] 任秀宝[2] 刘虹[2] 

机构地区：[1]中国科学院沈阳应用生态研究所,沈阳110016 [2]天津市肿瘤医院免疫室,天津300060

出　　处：《生物工程学报》2003年第6期750-753,共4页Chinese Journal of Biotechnology

基　　金：沈阳应用生态研究所与屹昌科技集团股份有限公司合作项目~~

摘　　要：采用酶切连接和重叠PCR连接两种方法将抗黑色素瘤单链抗体基因和去除N端信号肽的金黄色葡萄球菌肠毒素A基因进行融合 ,并将融合基因克隆于pET2 8 a表达载体上 ,转化大肠杆菌BL2 1(DE3)。用Ni NTA系统对表达产物进行分离、纯化。MTT法检测融合蛋白对黑色素瘤细胞的体外抑制率。结果表明 6His ScFv SEA融合蛋白可在E .coliBL2 1(DE3)中稳定表达 ,表达量占菌体蛋白的 30 % ,主要以包涵体的形式存在。融合蛋白可通过激活效应细胞对表达相关抗原的黑色素瘤细胞发挥抑制作用。Two strategies, direct ligation after enzyme digestion and over-lap PCR technology, were adopted to construct a fusion gene which was composed of the antimelanoma single chain antibody gene and the staphylococcal enterotoxin A gene without N-terminal signal sequence. The fusion gene was subcloned into pET28-a vector and transformed into E. coli BL21(DE3). Ni-NTA system was selected to separate and purify the expresstd products. The inhibition ratio of the fusion protein was tested by MTT method. It is shown that the 6His-ScFv-SEA fusion protein can be expressed stably in E. coli BL21 (DE3). The quantity of the fusion protein was shown up to 30% of the total protein of the bacteria and mainly in inclusion body. By activation the effective cells,the fution protein can inhibit the melanoma cell whith expressed corresponding antigen.

关 键 词：抗黑色素瘤单链抗体 金黄色葡萄球菌肠毒素A 融合蛋白 靶向治疗 基因表达 

分 类 号：Q786[生物学—分子生物学]