金黄葡萄球菌野生株肠毒素B重组蛋白的表达及纯化  

Expression and Purification of Recombinant Enterotoxin B Protein of A Wild Staphylococcus aureus Strain

作　　者：高世同[1] 张仁利[1] 刘会娟[2] 秦莉[1] 耿艺介[1] 黄达娜[1] 吴少庭[1] 

机构地区：[1]深圳市疾病预防控制中心医学分子生物室,深圳518020 [2]中山大学医学院达安基因诊断中心,广州510089

出　　处：《中国生物制品学杂志》2008年第10期865-868,共4页Chinese Journal of Biologicals

基　　金：深圳市重点医学实验室专项基金(2007).

摘　　要：目的在大肠埃希菌中表达金黄葡萄球菌野生株肠毒素B(SEB)重组蛋白,并进行纯化。方法采用PCR方法从金黄葡萄球菌基因组中扩增出SEB基因片段,与pMD18-T载体连接,构建重组质粒pMD18-T/SEB,测序分析后,亚克隆入表达载体pGEX-4T-2,转化感受态E.coliJM109,IPTG诱导表达。表达产物采用B-PER GST Fusion Protein Purification Kit纯化后进行Western blot鉴定。结果SEB基因扩增片段大小为705bp,测序结果显示与金黄葡萄球菌S6株序列相比无碱基的插入或缺失,同源性为98.4%,存在11个碱基变异,其中7个为无义突变,4个为有义突变(突变位点位于第364、699、899和988位,编码氨基酸变化分别为:Ser→Ala、Gln→His、Asn→Ser和Met→Leu);表达的含GST的融合蛋白相对分子质量约53000,纯化后经SDS-PAGE分析,可见单一条带。Western blot显示表达产物可被兔抗SEB抗体所识别。结论已成功表达并纯化了金黄葡萄球菌野生株SEB重组蛋白,为开发SEB免疫诊断试剂提供了材料。Objective To express recombinant enterotoxin B （SEB） protein of a wild Staphylococcus aureus strain in E. coli and purify the expressed product. Methods Amplify SEB gene fragment from the genome of wild S. aureus strain S704030 by PCR and insert into vector pMD18-T. Identify the constructed recombinant plasmid pMD18-T / SEB by sequencing and subclone into expression vector pGEX-4T-2. Transform the constructed recombinant plasmid pGEX-4T-2-SEB to competent E. coli JM109 for expression under induction of IPTG. The expressed product was purified by B-PER GST Fusion Protein Purification Kit and identified by Western blot. Results The amplified SEB gene, at a length of about 705 bp, showed a homology of 98. 4% to that of S. aureus strain S6. Compared with that of S6 strain, the amplified SEB gene showed no insertion or deletion of base. However, 11 base mutations were observed, of which 7 were nonsense mutations, and the other 4 were sense mutations at sites 364, 699, 899 and 988, resulting in the changes of amino acids from Ser to Ala, Gln to His, Asn to Ser and Met to Leu respectively. The expressed fusion protein containing GST, with a relative molecular mass of about 53 000, showed a single band on SDS-PAGE profile and was recognized by rabbit anti-SEB antibody as proved by Western blot. Conclusion The recombinant SEB protein of wild S. aureus strain S704030 was successfully expressed and purified, which provided materials for the development of diagnostic kit for SEB.

关 键 词：金黄葡萄球菌 肠毒素B 表达 纯化 

分 类 号：R378.11[医药卫生—病原生物学] Q786[医药卫生—基础医学]