点突变SEA(D227A)的基因构建、原核表达与鉴定

目的：进行点突变的SEA(D227A)基因原核表达载体的构建，并进行表达、分离纯化与鉴定。方法：采用PCR技术从产SEA的葡萄球菌标准株中克隆SEA基因，通过下游引物上的点突变，使得到的SEA基因752位碱基突变由A突变为c，致使SEA成熟蛋白的第227位氨基酸残基由D(DAT)突变为A(GCF)，构建pRSET：SEA(D227A)重组表达质粒，在大肠杆菌BL21(DE3)pLysS中进行原核表达，再通过Western blot进行鉴定。结果：构建的SEA(D227A)基因经测序证实与设计的序列一致。成功地构建pRSET-SEA(D227A)重组表达质粒，转化感受态大肠杆菌BL21(DE3)pLysS，通过IPTG诱导得到分子量约为32kD的蛋白，Western blot显示其能够与抗SEA的单克隆抗体特异性结合，最后采用Ni^2 -NTA柱进行分离纯化。结论：成功地构建SEA(D227A)基因原核表达载体，并在大肠杆菌中得到高效表达，为寻找有效低毒副作用的超抗原提供了线索。

Construction,prokaryotic expression and identification of mutant SEA(D227A)

Objective:To construct the prokaryotic expression vector of SEA mutant gene SEA(D227A).The gene was expressed in E.Coli, and the induced protein was purified and identified.Methods:The SEA gene was cloned by PCR from Staphylococcus aureus strain FRI 100.D227A was introduced by changing the Asp codon GAT into GCT(Ala)in the primer.The expression plasmid pRSET SEA(D227A)was constructed and transformed into E.Coli BL21(DE3)pLysS.The induced protein was identified by Western blot.Results:The nucleotide sequence of SEA(D227A)was found to be identical to the designed sequence. E.Coli BL21(DE3)pLysS contained pRSET SEA(D227A)can express a 32 kD protein which can specially bind with the anti SEA mAb.The induced protein was purified with Ni 2 system.Conclusion:The SEA(D227A)gene was constructed and expressed successfully.The study gave a clue to the research of low toxic superantigen.