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目的在大肠埃希菌中融合表达金黄色葡萄球菌(Staphylococcus.aureus,S.aureus)GapC蛋白与鼠伤寒沙门菌鞭毛蛋白,并检测其甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)活性。方法采用PCR技术分别扩增鼠伤寒沙门菌鞭毛蛋白flic基因和S.Aureus GapC基因,通过overlap PCR将这两段基因拼接并克隆至载体pQE30a,构建重组表达质粒pQE30-flic-GapC,将重组表达质粒转化E.coli XL-blue,IPTG诱导表达,表达产物经镍柱亲和层析试剂盒纯化后,进行SDS-PAGE及Western blot分析,并检测其GAPDH活性。结果重组表达质粒pQE30-flic-GapC经双酶切及测序鉴定证明构建正确;表达产物相对分子质量约95 000,最佳诱导表达时间为5 h,主要以包涵体形式存在,占菌体总蛋白的31.4%;纯化产物可与小鼠抗S.aureus全菌体多抗血清和抗鼠伤寒沙门菌全菌体多抗血清发生特异性反应,GAPDH活性为(0.337±0.019)。结论成功表达并纯化了具有较高生物学活性的flic-GapC融合蛋白,为S.aureus亚单位疫苗的研究奠定了基础。
Objective To express Staphylococcus aureus (S. aureus) GapC protein and Salmonella typhimurium flagellin in Escherichia coli and detect its glyceraldehyde-3-phosphate dehydrogenase dehydrogenase, GAPDH activity. Methods The flic and S.Aureus GapC genes of Salmonella typhimurium were amplified by polymerase chain reaction (PCR). The two genes were spliced and cloned into vector pQE30a by overlap PCR. The recombinant plasmid pQE30-flic-GapC was constructed, The plasmid was transformed into E.coli XL-blue and induced by IPTG. The expressed product was purified by nickel affinity chromatography kit and analyzed by SDS-PAGE and Western blot. The activity of GAPDH was detected. Results The recombinant plasmid pQE30-flic-GapC was confirmed by double enzyme digestion and sequencing. The recombinant plasmid pQE30-flic-GapC was constructed correctly. The relative molecular mass of the expressed product was about 95 000 and the optimal induction time was 5 h. The recombinant protein mainly existed in the form of inclusion bodies, 31.4%. The purified product reacted specifically with multiple antiserum against S.aureus and all antiserum against Salmonella typhimurium. The activity of GAPDH was (0.337 ± 0.019). Conclusion The flic-GapC fusion protein with high biological activity was successfully expressed and purified, which laid the foundation for the study of S.aureus subunit vaccine.