高级检索
杨浩民, 郁建平, 胡风庆. 金黄色葡萄球菌肠毒素C2基因定点突变分析[J]. 中国公共卫生, 2008, 24(11): 1334-1336. DOI: 10.11847/zgggws2008-24-11-30
引用本文: 杨浩民, 郁建平, 胡风庆. 金黄色葡萄球菌肠毒素C2基因定点突变分析[J]. 中国公共卫生, 2008, 24(11): 1334-1336. DOI: 10.11847/zgggws2008-24-11-30
YANG Hao-min, YU Jian-ping, HU Feng-qing. Site-directed mutagenesis of staphylococcal enterotoxin C2 His118[J]. Chinese Journal of Public Health, 2008, 24(11): 1334-1336. DOI: 10.11847/zgggws2008-24-11-30
Citation: YANG Hao-min, YU Jian-ping, HU Feng-qing. Site-directed mutagenesis of staphylococcal enterotoxin C2 His118[J]. Chinese Journal of Public Health, 2008, 24(11): 1334-1336. DOI: 10.11847/zgggws2008-24-11-30

金黄色葡萄球菌肠毒素C2基因定点突变分析

Site-directed mutagenesis of staphylococcal enterotoxin C2 His118

  • 摘要: 目的 利用金黄色葡萄球菌肠毒素C2(SEC2)基因His118定点突变,以获得超抗原性保持不变的减毒肠毒素。方法 利用PCR介导的定点突变技术,对SEc2基因中的His118密码子进行定点突变,得到的带有突变基因的表达质粒在大肠埃希菌中用异丙基硫代-β-D-半乳糖苷(IPTG)诱导其表达。采用镍离子金属整合(Ni-NTA)亲和层析和凝胶过滤层析纯化突变体蛋白,并测定突变体蛋白超抗原性及体外抑瘤效果。结果 应用点突变试剂盒,PCR扩增得到SEc2突变体表达质粒pET-28a-SEC2(H118Y)在1mmol/LIPTG诱导下高效表达。突变体蛋白SEC(H118Y)在2~200 ng时刺激人体细胞增殖效果与SEC2基本一致,体现二者具有相同的超抗原性。SEC(H118Y)时,在2~200 ng与SEC2抑制大肠癌细胞Cx-1效果基本相同,而在500 ng时,SEC(H118Y)抑瘤效果明显优于与SEC2。结论 SEC(H118Y)超抗原性和抑瘤活性没有因His118位点的突变而改变。

     

    Abstract: Objective To obtain staphylococcal enterotox in(SE)without any side-efffects by selecting His118 of SEc2 as substitutional site through site-directed mutagenesis so as to preserve superantigenic activity.Methods His118 was substituted through PCR.The resultant plasmid harboring the mutated SEc2 was expressed in E.coli under the existance of IPTG.The proteins purified through the Ni-NTA affinity chromatography and Gel filtration chromatography were used to analyze superantigenic activity and anti-tumor effect.Results Site-directed mutagenesis was perfo rmed using MutanBEST kit through PCR.The resultant plasmid pET-28a-SEC2(H118Y)was efficiently expressed in Escherichia coli with 1 mMIPTG.The purified SEC(H118Y)exhibited the same superantigenic activity as SEC2 according to the results of PBMC proliferation activity when the dose was between 2 ng and 200 ng.SEC(H118Y)also exhibited the same anti-tumor effect as SEC2 when the dose was between 2 ng and 200 ng.The anti-tumor effect of SEC(H118Y)was better than SEC2 when the dose was 500 ng.Conclusion A lthough His118 of SEC2 was mutated,superantigenic activity and anti-tumor effect of SEC(H118Y)was not changed.

     

/

返回文章
返回