【摘要】 利用PCR从金黄色葡萄球菌标准株 (Staphylococcusaureus,ATCC13 5 65 )中克隆了金黄色葡萄球菌肠毒素A(SEA)的基因 ,序列测定结果与报道完全一致。构建了表达载体pET SEA并获得高效表达 ,重组蛋白 (rSEA)在 3 7℃诱导时以包涵体形式存在 ,降低温度则出现可溶表达 ,可溶性rSEA占总rSEA的 5 5 %。可溶性rSEA经Ni2 + 亲和层析纯化 ,达电泳纯。通过同源模建对rSEA对SEA进行结构比较 ,结果表明尽管rSEA比野生型SEA多了 9个氨基酸但其结构并没有明显的变化。单核细胞增殖试验进一步证明了该结论 :将rSEA与SEA同外周血单个核细胞共同培养 ,两者均能有效地促进其增殖。将rSEA与体内激活的脾细胞共培养 ,则能增强脾细胞的体外抑瘤活性更多还原

【Abstract】 The superantigen,such as staphylococcal enterotoxins, had been identified as possible anti-cancer molecules in many reports. In this paper, we cloned the entA gene encoding Staphylococcal enterotoxin A from the genomic DNA of Staphylococcus aureus(ATCC13565) by PCR, the sequence cloned was accordance with that reported in Genebank .The entA gene could be expressed effectively after inserted into plasmid pET-22b(+), The rSEA was expressed as inclusion bodies when induced by IPTG at 37 ℃ and became soluble after induced at low temperature,the soluble part is about 55% of total rSEA products .Only one band was detected by western-blotting in expression product of BL-21(DE3) with pET-SEA. The soluble rSEA was purified by Ni 2+ chelating sepharose column .No other protein except rSEA was seen in SDS-PAGE gel stained by both Coomassie brilliant blue and silver salt, which showed that the rSEA was purified effectively. Homology modeling of rSEA determined the structure change was conducted , which indicated there was no apparent structure change between rSEA and native SEA. This result was also confirmed by proliferation assay of PBMC, for the rSEA could induced proliferation of PBMC as effectively as native SEA . The increasing anti-tumor activity of rSEA was also detected after the spleen cell activated in vivo by rSEA ,which was accordance with others reports. This work paved the way for the further study of anti-cancer with rSEA.更多还原