葡萄球菌肠毒素O表达及其生物学活性的初步分析  

Expression and bioactivity of staphylococcal enterotoxin O

作　　者：刘莹[1] 黄金海[1] 孙跃辉[1] 张丽霞[1] 王建华[2] 庄世文[1] 王静思[1] 李琳[1] 徐丹丹[1] 

机构地区：[1]天津大学农业与生物工程学院,300072 [2]天津市出入境检验检疫局

出　　处：《中华微生物学和免疫学杂志》2009年第12期1094-1098,共5页Chinese Journal of Microbiology and Immunology

基　　金：天津市应用基础及前沿技术研究计划项目（08JCZDJC22600）

摘　　要：目的克隆葡萄球菌肠毒素O（seo）基因，构建其两种原核表达载体，表达、纯化重组蛋白SEO，并对其生物活性进行初步研究。方法设计引物PCR获得seo成熟肽基因，将其分别克隆至原核表达载体pGEX-6P-1、pET28a，转化E．coli BL21、E．coli BL21（DE3），重组蛋白分别经GST亲和层析、镍螯合层析纯化，利用Westernblot验证重组SEO的免疫原性，MTT法测定重组SEO对ICR小鼠脾淋巴细胞的增殖作用，Caspase3酶活及DNA电泳分析重组SEO诱导细胞凋亡的能力。结果克隆的seo基因序列与报道的序列完全相同。纯化获得重组蛋白GST—SEO和P28-SEO的表达量分别占菌体蛋白的25％和22％。免疫印迹表明两种蛋白均具有良好的免疫原性。生物活性检测表明，低剂量的肠毒素对小鼠脾淋巴细胞有刺激增殖作用，较高剂量则会导致细胞凋亡。结论两种重组的SEO仍具有超抗原的活性，对小鼠脾淋巴细胞具有诱导凋亡的能力。Objective To clone the mature peptide gene of seo, construct two kinds of prokaryotic fusion expression vector, obtain recombinant staphylococcal enterotoxin O（SEO） and evaluate its biological activity. Methods The seo gene was amplified from ZNZ2-3 strain of Staphylococcus xylosus by designed primers and it was cloned into fusion vector pGEX-6P-1 and pET28a. The soluble recombinant protein（ GST- SEO） was expressed in E. coli BL21 host cells, the purified GST-SEO was obtained by a single step of affinity chromatography using Glutathinione Sepharose 4B colume. Another fusion protein（P28-SEO） with his-tag was expressed in E. coli BL21 （ DE3 ） , the purified P28-SEO protein was harvested by nickel chelate affinity chromatography method. The antigenicity of recombinant SEO was tested by Western blot. The stimulating effects of recombinant SEO on mouse lymphoeytes was tested by MTT. The activity of Caspase 3 and the apoptosis of DNA ladder were tested. Results The nucleotide sequence of the cloned seo gene was the same as that of reported in GenBank. The soluble recombinant protein GST-SEO was expressed at 25% expression level, and P28-SEO was 22%. The Western blot by GST-SEO-positive sera demonstrated that the recombinant SEO had good immunogenicity. The MTT assay of the enterotoxin activity showed that low dose of recombinant SEO stimulated the proliferation of mice spleen lymphoeytes, higher doses will lead to apoptosis. Conclusion Two of the recombinant SEO still have superantigen activity, and can lead to apoptosis of mice splenocytes.

关 键 词：葡萄球菌 肠毒素O 生物学活性 

分 类 号：R686[医药卫生—骨科学]