中国人兽共患病杂志2004，Vol.20Issue(4)：303-306,310,5.
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
Construction of eukaryotic expression system of staphylococcal enterotoxin A gene and identification of the target recombinant protein
摘要
Abstract
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 ampli fied target fragment was sequenced after T-A cloning. The pUCm-T-SEA gene fragment obtained by endonuclease digestion was linked to the eukaryofic expression vector pPIC9K, and the recombinant eukaryotic vector pPIC9k-SEA was transformed into P. pastoris GS115 strain by means of electrofnsion 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 induetion 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.关键词
Staphylococcal enterotoxin A/SEA gene/cloning/eukaryotic expression system/construction/recombinant protein/expressionKey words
Staphylococcal enterotoxin A/SEA gene/cloning/eukaryotic expression system/construction/recombinant protein/expression分类
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孙爱华,王邵浙新,王阮萍,毛亚飞,严杰..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[J].中国人兽共患病杂志,2004,20(4):303-306,310,5.基金项目
This subject was supported by Science Reseach Plan of Zhejiang Province (No. :2003C34010) （No. :2003C34010）
