葡萄球菌肠毒素B基因真核表达系统的构建及目的蛋白的鉴定

目的　克隆葡萄球菌肠毒素B(SEB)基因 ,构建其真核表达系统并表达目的蛋白。方法　采用高保真PCR从金黄色葡萄球菌FRIS6B株中扩增全长SEB ,目的片段T A克隆后测序。重组质粒pUCm T SEB经酶切获得的pUCm T SEB基因片段与真核表达载体pPIC9K连接。将重组真核载体pPIC9K SEB转化入P .pastorisGS115株 ,经PCR筛选并鉴定pPIC9K SEB P .pastorisGS115。在 0 5 % (v v)甲醇诱导下 ,采用SDS PAGE检测重组SEB(rSEB)的表达 ,并对rSEB的N端氨基酸序列和诱导人脐静脉内皮EVC 30 4细胞分泌IL 1α和TNFα的作用进行了检测。结果　所克隆的SEB基因核苷酸及氨基酸序列的同源性分别高达 98 84 %～ 10 0 %和 10 0 %。表达的SEB在SDS PAGE图谱的位置与预期相符。rSEB氨基末端氨基酸序列与预期完全相符。rSEB与SEB商品有相似的促人脐静脉内皮细胞EVC 30 4分泌IL 1α和TNFα的活性。结论　已成功地构建了SEB的真核表达系统 ,为进一步分析SEB分子中毒性相关活性位点、定位突变获得减毒或无毒的突变体奠定了基础。

Construction of Eukaryotic Expression System of Staphylococcal Enterotoxin B Gene and Identification of Goal Protein

Objective To clone staphylococcal enterotoxin B (SEB) gene and construct an eukaryotic vector expressing the gene. Methods Amplify the full-length of SEB gene from FRIS6B strain of Staphyloccus aureus by high fidelity PCR,insert into pUCm-T vector by T -A cloning,subclone into eukaryotic expression vector pPIC9K and transform to G S115 strain of P.pastoris to construct an eukaryotic expression system pPIC9 K-SEB-P.pastoris GS115.Express recombinant SEB(rSEB) under induction of 0 .5% (v/v) methanol and identify the expressed product by SDS-PAGE and N-termi nal sequencing.The expressed rSEB was used to induce the secretion of I L-1α and TNFα from human umbilical vein endothelial cell EVC-304.Res ults The homologies of nucleotide and amino acid sequences of the clone d SEB gene to those reported in Genbank were 98.84%-100% and 100% respectively .Both the relative molecular weight shown on SDS-PAGE profile and the sequence at N-terminal of rSEB gene we re consistent with those expected.No significant difference was observed between rSEB and commrcial SEB in activity of promoting the secretion of IL-1α and TN Fα by human umbilical vein endothelial cell EVC-304. Conclusion The successful construction of eukaryotic expression system of SEB laid a foundation of analyzing the toxicity-related activity site in SEB molecule and obtaining attenuated or avirulent mutant by site-directed mutatio n.