金黄色葡萄球菌肠毒素B基因在大肠杆菌中的克隆及表达  

Cloning and expression of Staphylococcus aureus enterotoxin B gene in Escherichia coli

作　　者：李飞[1,2] 周帅[1,2] 董慧[1,2] 黄艳梅[1,2] 梁秉绍 李娟[1,2] 龙燕[1,2] 王洁琳[1,2] 谢永强[1,2] 杨镒宇[1,2] 周珍文[1,2] 

机构地区：[1]广州医科大学,广州510120 [2]广州市妇女儿童医疗中心,510120

出　　处：《检验医学与临床》2016年第18期2553-2555,共3页Laboratory Medicine and Clinic

基　　金：广州市医药科技重点项目(201102A212013);广东省科技厅项目(2014A020212013)

摘　　要：目的扩增金黄色葡萄球菌肠毒素B(SEB)基因,构建其原核表达载体,并进行诱导、表达,为其应用研究奠定基础。方法根据GenBank中SEB的基因序列,设计一对分别含BamHⅠ、XhoⅠ酶切位点的特异性引物,以金黄色葡萄球菌基因组DNA为模板进行PCR扩增后,经BamHⅠ、XhoⅠ双酶切,并与做相应酶切的pET-28α(+)连接,转化大肠杆菌BL21,提取质粒进行双酶切鉴定及测序,用IPTG诱导表达融合蛋白,SDS-PAGE和Western blot印迹鉴定表达产物。结果成功扩增出SEB基因,基因大小为801bp,重组PET-28α(+)-SEB双酶切鉴定可见目的片段,测序结果显示SEB在正确读框中,序列比对分析显示其与相关报道核苷酸序列一致性达99%。经IPTG诱导后,pET-28α(+)-SEB/BL21在相应的相对分子质量(35×103)可见融合蛋白以包涵体形式表达,免疫印迹在相应分子量检测到目的蛋白。结论克隆了SEB基因,并成功在大肠杆菌BL21中以包涵体形式表达,为肠毒素B应用研究奠定了基础。Objective To amplify the Staphylococcus aureus enterotoxin B （SEB） gene and to construct its prokaryotic expres‐sion vector for conducting induction and expression to lay the foundation for its application research .Methods According to SEB gene sequences in the GenBank ,a pair of specific primer containing BamH Ⅰ and Xho Ⅰ enzyme digestion sites were designed .The SEB genome DNA served as the template for conducting PCR amplification ,then after BamH Ⅰ and Xho Ⅰ dual enzyme digestion , connected with the pET‐28α（＋ ） for making corresponding enzyme digestion .Escherichia coli BL21 was transformed .The plasmids were extracted for conducting the dual enzyme digestion identification and sequencing .The fusion protein was expressed by using the IPTG induction .The expressed product was identified by Western blot .Results SEB gene was successfully amplified ,which size was 801bp ,the recombinant PET‐28α（＋ ）‐SEB dual enzyme digestion identification could find the target fragments ,the sequen‐cing results displayed that SEB was in the correct reading frame ,the sequencing comparative analysis showed that its consistency to nucleotide sequence in the related report was 99% .After IPTG induction ,the fusion protein could be expressed as the inclusion body in the corresponding molecular weight 35 × 103 of pET‐28α（＋ ）‐SEB/BL21 .The target protein could be detected in the corre‐sponding molecular weight by Western blot .Conclusion SEB gene is cloned and successfully expressed as the inclusion body in Escherichia coli BL21 ,which lays a foundation for staphylococcus aureus SEB application research .

关 键 词：金黄色葡萄球菌 肠毒素B 克隆 表达 

分 类 号：R155.31[医药卫生—营养与食品卫生学]