齿垢密螺旋体精氨酸激酶TDE2037的原核表达与纯化  

Prokaryotic expression and purification of an arginine kinase gene TDE2037 of Treponema denticola

作　　者：陈宇星[1,2] 周鹏 唐路 陈文霞 

机构地区：[1]广西医科大学口腔医学院牙体牙髓科,南宁530021 [2]美国阿拉巴马大学伯明翰分校牙学院儿牙系,伯明翰al35205 [3]首都医科大学宣武医院口腔科,北京100053

出　　处：《广西医科大学学报》2017年第12期1686-1690,共5页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(No.81160133)

摘　　要：目的:构建齿垢密螺旋体TDE2037基因的原核表达质粒,表达及纯化TDE2037精氨酸激酶蛋白。方法:以齿垢密螺旋体基因组DNA为模板,PCR扩增TDE2037基因,PCR产物经限制性内切酶消化后,由T4 DNA连接酶连接原核表达载体pET-21a以及pET28a-SUMO,连接产物分别转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达目的蛋白,采用镍柱亲和层析法纯化目的蛋白,并使用分子排阻预装柱提高蛋白纯度。结果:成功构建TDE2037基因的原核表达质粒,经测序与基因库(Genbank)的TDE2037序列一致,在大肠杆菌BL21(DE3)中成功诱导表达目的蛋白,融合蛋白由pET-21a-TDE2037表达形成包涵体;由pET28a-TDE2037-SUMO表达部分形成包涵体,部分为可溶蛋白。镍柱亲和层析法成功纯化目的蛋白。结论:成功构建齿垢密螺旋体TDE2037基因的原核表达质粒,并表达纯化了重组融合目的蛋白,为下一步研究奠定基础。Objective： To construct the prokaryotic expression vectors of Treponema denticola gene TDE2037,and express and purify. Methods： TDE2037 was amplified by PCR and inserted into pET-21 a and pET28 a-SUMO plasmids. Recombination plasmids were transformed into E.coli BL21 to express recombined protein under induction of IPTG. The expressed products were identified by biological mass spectrometry and the targeted protein was purified with metal affinity chromatography column. Results： TDE2037 was amplified successfully and cloned into pET-21 a and pET28 a-SUMO plasmids. Sequences were identified to de consistent with the data of Genbank. Recombination proteins were successfully expressed in E.coli BL21. The protein of pET-21 aTDE2037 completely existed in form of inclusion body and remained insoluble,whereas the expression product of pET28 a-TDE2037-SUMO,partially soluble and partially in form of inclusion body. High purity protein was achieved by metal affinity chromatography column. Conclusion： The prokaryotic expression vectors of Treponema denticola gene TDE2037 were successfully constructed and the expressed protein was successfully purified,which may lay a basis for the further study of TDE2037.

关 键 词：齿垢密螺旋体 蛋白纯化 精氨酸激酶 

分 类 号：Q51[生物学—生物化学]