葡萄球菌肠毒素A基因真核表达系统的构建及其目的重组蛋白的鉴定(英文)

Construction of eukaryotic expression system of staphylococcal enterotoxin A gene and identification of the target recombinant protein

目的 克隆金黄色葡萄球菌肠毒素A(SEA)基因、构建其真核表达系统及目的重组蛋白的表达情况。方法 采用高保真PCR从金黄色葡萄球菌ATCC135 6 5菌株中扩增全长SEA ,目的扩增片段T -A克隆后测序。经核酸内切酶酶切重组质粒 pUCm -T -SEA获得的 pUCm -T -SEA基因片段与真核表达载体 pPIC9K连接。采用电融合法将重组真核载体pPIC9K -SEA转化入P pastorisGS115株 ,构建成真核表达系统 pPIC9K -SEA -P pastorisGS115。采用含G4 18抑制剂的YPD琼脂平板和PCR筛选并鉴定 pPIC9K -SEA -P pastorisGS115。 0 5 % (V∶V)甲醇诱导下 ,采用SDS -PAGE检查BMMY液体培养基上清中重组SEA(rSEA)的表达情况。结果 可从S aureusATCC135 6 5株DNA中扩增获得SEA目的片段。与报道的SEA基因序列 (GenBankNo :AP0 0 4 82 8andL2 2 5 6 6 )比较 ,所克隆的SEA基因核苷酸及氨基酸序列的相似性分别高达 98 84 %～ 10 0 %和 10 0 %。在甲醇的诱导下 ,pPIC9K -SEA -P pastorisGS115能表达在SDS -PAGE图谱位于预期位置的rSEA。结论 我们成功地构建了能表达SEA的真核表达系统 ,为进一步分析SEA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。

To clone the staphylococcal enteroloxin A (SEA) gene and to construct the eukaryotic expression system and the target recombinant proteins,the entire SEA gene from strain of ATCC 13565 DNA was amplified by high fidelity PCR and the amplified target fragment was sequenced after T-A cloning. The pUCm-T-SEA gene fragment obtained by endonuclease digestion was linked to the eukaryotic expression vector pPIC9K,and the recombinant eukaryotic vector pPIC9k-SEA was transformed into P.pastoris GS115 strain by means of electrofusion method,thus to consruct an eukaryotic expression system pPIC9k-SEA-P.pastoris GS115.This eukaryotic expression system was screened and identified by using YPD agar plates containing inhibitor G418 and PCR.The expression of recombinant SEA(rSEA) in the supermatants of BMMY liquid medium was determined by SDS-PAGE with induction of 0.5%(vv) of methanol.It was found that the target DNA fragment of SEA gene with expected size could be amplified from strain ATTCC13565 of Staphylococcus aureus.In comparison with the gene sequences reported (GenBank No.AP004828 and L22566),the simiarities in nucleotides and amino acids of the SEA gene cloned were high up to 98.84%-100% and up to 100% respectively. This eukaryotic expression system was able to express the recombinant SEA under the induction with 0.5% of methanol at that location of the expected position in SDS-PAGE analysis.It concludes that a recombinant eukaryotic expression system to express SEA gene is successfully constructed in this study,and this would establish a foundation for the further analysis of the toxicity-associated activities of SEA or to obtain the detoxified or non-toxic mutants.