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

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。

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

Objective: To enhance the anti-tumor activity of staphylococcal enterotoxin,His 31 of ataphylococ cus 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.