沙眼衣原体主要外膜蛋白T、B细胞多表位基因的表达及其鉴定

Expression and identification of T cell and B cell multi-epitopes from major outer membrane protein of Chlamydia trachomatis

目的 对沙眼衣原体（Ct）主要外膜蛋白（MOMP）细胞毒性T淋巴细胞（CTL）表位进行预测和选择，并进行Ct MOMP多表位基因克隆和多表位蛋白的原核表达及其抗原性分析，为研制多表位Ct疫苗提供基础资料。方法利用SYFPEITHI法和多项式方案结合预测HLA-A2限制性的0MOMP的CTL表位，选取含多个CTL表位的基因片段作为目的基因，兼顾目的基因上下游存在的TH和B细胞表位的基因，共同组成含CTL、TH和B细胞表位的多表位基因。多表位基因经密码子优化后进行全序列合成，克隆入原核表达载体pET-32a（＋），经IPTG诱导在大肠杆菌BL21（DE3）表达并纯化，经SDS-PAGE和Western blot分析鉴定表达产物。结果 经预测筛选得到了多个MOMPHLA-A2限制性的CTL表位，成功克隆了含CTL、TH、B表位的MOMP多表位基因，并在大肠杆菌中获得了高效表达。表达产物的相对分子质量（Mr）约24×10^3，与预期肘，相符，并用Western blot方法初步证实多表位蛋白有抗原特异性。结论 成功设计了Ct MOMP的T、B细胞多表位基因，并证实在原核表达系统中获得正确表达的多表位蛋白具有良好的抗原性。

Objective To identify the potential muhi-epitopes vaccine of Chlamydia trachomatis （Ct）. Methods Long-distance prediction system SYFPEITHI and polynomial method were used to predict the HLA-A2 restricted CTL epitopes of Ct-MOMP. The muhi-epitopes gene was then cloned into the prokaryotic expression vector pET-32a （ ＋ ） after synthesizing and then expressed in E. coli BL21 （DE3）. After induction with IPTG, the expressed multi-epitopes protein of MOMP was purified by Ni-NTA affinity chromatography and identified by SDSPAGE and Western blot. Results The HLA-A2 restricted CTL muhi-epitopes of Ct MOMP were predicted and the gene of Ct MOMP including CTL, TH and B cell epitopes was cloned and expressed in prokaryotic expression system. SDS-PAGE analysis showed the relative molecular mass of tiffs muhi-epitopes protein as 24 × 10^3 , which was consistent with the that of theoretically predicted, and the specificity of this muhi-epitopes protein was confirmed by Western blot. Conclusion The multi-epitopes gene of Ct MOMP was successfully cloned and expressed in prokaryotic expression system, and the results confirm that multi-epitopes protein has certain specificity.