摘要
目的在原核质粒中克隆金黄色葡萄球菌(S.aureus)Esx A基因,并进行表达及纯化,为其功能研究奠定基础。方法根据Gene Bank中S.aureus Esx A序列,设计特异性引物PCR扩增Esx A基因,PCR产物纯化后经Eco RΙ和XhoΙ双酶切,连接至p ET-28a(+)载体,转化大肠杆菌BL21(DE3),IPTG诱导重组蛋白表达,His柱纯化重组Esx A蛋白,SDS-PAGE和Western blot验证重组蛋白的表达。结果成功克隆Esx A基因,基因全长294 bp,起始于ATG,终止于TAA,预测的分子量为11 036.2,等电点为4.61,经双酶切在294 bp处可见目的条带,基因测序显示Esx A在正确阅读框内,提示重组蛋白构建成功。SDS-PAGE显示重组蛋白在分子量大约14.5×103 Mr处左右见到可溶性的蛋白表达,Western blot分析识别14.5×103 Mr重组蛋白。结论在大肠杆菌中成功克隆表达Esx A蛋白,为其功能研究奠定基础。
Objective To clone Esx A gene of Staphylococcus aureus（ S.aureus）, express and purify recombinant Esx A protein in prokaryotic system,and lay the foundation for the research of its function. Methods Specific primer of Esx A gene was designed according to Gene Bank Esx A sequence. PCR products were digested by Eco RΙ and XhoΙ,then ligated to p ET-28a（＋）,followed by transformation to Escherichia coli BL21（DE3）. Recombinant protein expression was induced by IPTG and purified by His column. Recombinant protein was identified by SDS-PAGE and Western blot analysis.Results Esx A gene was cloned; the length was 294 bp; the sequences started with ATG and end with TAA. The predicted molecular weight was 11 036.2 and isoelectric point was 4.61.A 294 bp band was obtained by recombinant plasmid double digestion. SDS-PAGE showed that the recombinant protein weight was about 14.5×103Mr. Western blot analysis identified a 14.5 ×103Mr recombinant protein. Conclusion Esx A was cloned and expressed in E. coli, which laid the foundation for the research of its function.
出处
《热带医学杂志》
CAS
2015年第6期728-730,740,共4页
Journal of Tropical Medicine
基金
广州市医药科技重点项目(201102A212013)
