葡萄球菌肠毒素C2突变体的构建及其体外抗肿瘤活性分析  

Construction and In Vitro Anti-Tumor Activity Analysis of Staphylo coccus Enterotoxin C2 Mutant

作　　者：王君[1,2] 郑玉玲[1] 郑欣[2] 谷丽维[3] 陈思维[2] 江华[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071 [2]沈阳药科大学生命科学与生物制药学院,辽宁沈阳110016 [3]沈阳药科大学中药学院,辽宁沈阳110016

出　　处：《生物技术通讯》2012年第5期631-634,共4页Letters in Biotechnology

基　　金：国家科技重大专项(2009ZX09103-618)

摘　　要：目的：利用基因定点突变的方法将葡萄球菌肠毒素C2（SEC2）的31位定点突变,以获得抑瘤效果增强的肠毒素。方法：利用基因定点突变的方法,将SEC2中31位的His用Asn替代,转入大肠杆菌中诱导表达,采用CM弱阳离子层析柱纯化蛋白,并用SDS-PAGE和Western印迹对其进行鉴定,通过MTS法检测其体外抗肿瘤活性。结果：构建了突变体蛋白SEC2（H31N）,并在大肠杆菌中实现了高效表达。体外实验表明,在相同浓度下,SEC2（H31N）对多种肿瘤细胞的抑制作用明显优于野生型SEC2,尤其在低浓度下此现象更为明显。对于5个受试细胞株,SEC2（H31N）的IC50均低于SEC2;SEC2（H31N）浓度分别为0.01~10、0.01和0.1 ng/mL时,对肿瘤细胞SMMC-7721、HepG2、A549的生长抑制率与SEC2相比均显著提高。结论：同野生型SEC2相比,突变体蛋白SEC2（H31N）对肿瘤生长的抑制作用得到了提高。Objective： To enhance the anti-tumor activity of staphylococcal enterotoxin, His 31 of ataphylococcus enterotoxin C2（SEC2） was substituted by Asn through site-directed mutagenesis. Methods： A superantigen SEC2（H31N） mutant was constructed by site-directed mutagenesis, and expressed in E.coli BL21（DE3）. The pro- tein was purified by CM ion-exchange chromatography and identified by SDS-PAGE and Western blot. MTS was used to analyze anti-tumor activity in vitro. Results： The mutated protein SEC2（H31N） was constructed and ex- pressed in E.coli efficiently. The MTS results showed that at the same concentration, the anti-tumor effect of SEC2 （H31N） was better than SEC2 on a variety of tumor cells in vitro, especially at the low concentrations. For five cell lines tested, the IC50 of SEC2（H31N） was all lower than that of SEC2. For SMMC-7721, HepG2 and A549, the tumor growth inhibition occurred at 0.01-10, 0.01 and 0.1 ng/mL of SEC2（H31N） respectively, which was sig- nificant lower than that of SEC2. Conclusion： Compared with the wild type SEC2, SEC2（H31N） mutant exhibited an enhanced anti-tumor response .

关 键 词：葡糖球菌肠毒素C2 超抗原 突变体 抗肿瘤 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]