【摘要】 目的:构建奈瑟淋球菌表面蛋白A(Neisseria surface proteinA,NspA)基因原核表达载体,并诱导NspA融合蛋白在宿主菌BL21(DE3)中表达。方法:根据基因库报道的淋球菌nsp A基因序列,设计合成一对引物;以淋球菌基因组DNA为模板,PCR扩增淋球菌Nsp A基因;将PCR扩增产物与带有谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)基因的高效原核表达质粒pGEX4T-1定向重组,构建原核表达重组体pGEX4T-1-NspA;重组体经酶切、PCR鉴定和核酸序列分析正确后,异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达,用十二烷基硫酸钠(SDS)-聚丙烯酰胺凝胶(PAGE)及Western blot技术检测重组蛋白表达情况。结果:重组体pGEX4T-1-Nsp A经BamHⅠ、XholⅠ双酶切、PCR和核酸序列分析表明:成功构建淋球菌Nsp A基因原核表达重组体pGEX4T-1-Nsp A;SDS-PAGE及Western blot分析显示:重组Nsp A基因在原核系统中得到了表达,融合蛋白GST-Nsp A约44 ku。结论:淋球菌Nsp A基因在大肠杆菌中得到了表达,为进一步研究淋球菌Nsp A蛋白免疫学性能和研制淋球菌疫苗提供了研究基础。更多还原

【Abstract】 Objective:To construct the prokaryotic expression system of Nsp A gene of Neisseria gonorrhoeae and induce it ex pressed in E.coli BL21(DE3).Methods:A pair of primers was designed according to the known sequence of Nsp A gene from GeneBank. The fragment of Nsp A gene was amplified from genomic DNA of Neisseria gonorrhoeae by polymerase chain reaction(PCR),and PCR product was cloned into pGEX4T-1 which containing GST gene to construct the prokaryotic expression system of pGEX4T-1-Nsp A.The recombinant plasmid of pGEX4T-l-Nsp A was analyzed with restriction endonuclease digestion,PCR and DNA sequencing analysis.The verified recombinant was transformed into E.coli BL21(DE3) and expressed by isopropyl-β-D-thiogalactoside (IPTG) induced.The fusion protein GST-Nsp A was examined with SDS-PAGE and Western blot.Results:The prokaryotic expression system of pGEX4T-l-Nsp A with Neisseria gonorrhoeae Nsp A gene was constructed successfully and verified by BamHⅠ,XholⅠrestriction endonuclease digestion,PCR and DNA sequencing analysis.By the analysis of SDS-PAGE and Western blot,the recombinant Nsp A(rNsp A) of Neisseria gonorrhoeae was expressed in prokaryotic expression system successfully.The fusion protein GST-Nsp A expressed is about 44 kDa.Conclusion;The Nsp A gene of Neisseria gonorrhoeae is expressed in E.coli, which provide a basis for the study immunological activity of the Nsp A protein of Neisseria gonorrhoeae and mucosal immune vaccine of Neisseria gonorrhoeae.更多还原