HSP-沙眼衣原体MOMP T细胞表位基因的重组、原核表达和蛋白质纯化

Gene recombinant and prokaryotic expression of HSP65-T cell epitopes of MOMP of C. trachomatis fusion protein and its purification

目的:构建热休克蛋白65(HSP65)与沙眼衣原体(C．trachomatis,Ct)主要外膜蛋白(MOMP) T细胞表位(简称ctml)融合蛋白(简称H-ctml)的原核表达载体,并将其表达及纯化。方法:用PCR技术获得HSP65基因和ctml基因,并分别将其克隆入pMD18-T载体,采用酶切与连接的方法从pMD18-T载体上获得HSP65基因片段,并将其克隆入pET28a表达质粒,再用相同的方法将ctml基因连接于HSP65基因片段的下游,从而完成H-ctml的基因重组。用IPTG诱导转化H—ctml表达载体的大肠杆菌,以镍金属螯合亲和层析法纯化融合蛋白质。结果:构建了HSP65与ctml的表达载体pET28a-H—ctml,DNA序列测定结果表明构建正确。以镍金属螯合亲和层析获得纯度为98％的融合蛋白质。结论:用分子克隆技术正确构建HSP65与 Ct MOMP T细胞表位融合蛋白的表达载体,并成功表达与纯化出具有生物活性的融合蛋白。

Objective To construct the prokaryotic expression vector of HSP65-T cell epitopes of MOMP of C. trachomatis （Ct） fusion protein （called H-ctml）, express and purify H-ctml. Methods The HSP65 and gene sequence of T cell epitopes of MOMP of Ct （ctml） were obtained by PCR method and cloned into pMD18-T vector respectively. The T vector was digested by endonuclease for releasing the fragment of HSP65 or ctml gene. The gene of HSP65 and ctml were cloned into pET28a plasmid successively to construct the prokaryotic expression vector （pET28a-H-ctml）, which recombined the gene of HSP65 and ctml. The E. cell BL21 （DE3） transformed with pET28a-H-ctm1 were induced by IPTE for expression of H-ctml fusion protein. The protein was purified by Ni2＋ affinity chromatography. Results The prokaryotic expression vector, pET28a-H-ctml, was constructed successfully. The fusion protein of H-ctml was expressed in E. cell BL21 （DE3）. The purity of fusion protein was 98% after purified by Ni^2＋ affinity chromatography. Conclusion The prokaryotic expression vector of pET28a-H-ctml has been constructed, and the fusion protein with biological activity has been successfully expressed and purified.