肺炎嗜衣原体主要外膜蛋白基因片段在大肠杆菌中的表达与鉴定

表达肺炎嗜衣原体（Chlamydia pneumoniae，Cpn）主要外膜蛋白（MOMP）的可变区VD2-VD3区．纯化产物并进行免疫活性分析，为探索重组蛋白在肺炎嗜衣原体血清学诊断中的应用提供资料．应用聚合酶联反应（PCR）技术，从肺炎嗜衣原体标准株AR-39的MOMP上扩增出抗原优势表住VD2-VD3区，将目的片段定向插入pET-30a载体，转化大肠杆菌B121，IPTG诱导表达并以Ni-NTA亲和层析柱纯化表达产物并行western-blot鉴定．成功构建了pET-30a-MOMPVD2-VD3的原核表达系统，表达并纯化出相对分子质量（Mr）为24×10^3Da的重组蛋白．Western-blot证实重组蛋白能与Cpn MOMP多克隆抗体发生特异性反应．肺炎嗜衣原体的MOMPVD2-VD3基因可以在大肠杆菌中得到表达，其表达产物能与相应的抗体结合，为肺炎嗜衣原体诊断候选抗原的研究奠定了基础．图4，参9．

Expression and Identification of Major Outer Membrane Protein of Chlamydia pneumoniae in Escherichia coli

To construct a recombinant vector eontainting the gene encoding immuno - dominant epitope of major outer membrane protein （omp） of Chlamydia pneumoniae（Cpn） , and to express and purify the recombinant protein, to find a new antigen for the exploiting diagnosis of C. pneumoniae. The immunodominant epitope of ompA gene was amplified from C. pneumoniae complete genome by polymerase chain reactions （PCR）, then the PCR product was directly cloned into pET30a vector to construct the pET30a - MOMPVD2-VD3 recombinant plasmid. After induction in E. coli BL21 （ DE3 ） by IPTG, recombinant protein MOMPVD2 - VD3 was expressed, then it was purified with Ni - NTA - his affinity chromatography and analyzed by Western blot. The 451bp targeted gene was successfully inserted into the prokaryotic expression vector pET30a. A recombinant protein with molecular weight about 24KDa was obtained after expression and purification and mainly existed in the pattern of inclusion body. Western blot proved that the recombinant protein can specifically react with C. pneumoniae MOMP PcAb. The expressed recombinant protein could specifically react with C. pneumoniae MOMP PcAb. The results lay the foundation for development of quick diagnostic kit applying to detect C. pneumoniae. 4figs. ,9refs.