沙眼衣原体质粒蛋白pORF5原核表达及免疫原性鉴定

Expression and immunogenicity of the pORF5 plasmid protein from Chlamydia trachomatis

目的构建沙眼衣原体(CT)pORF5蛋白基因重组质粒pGEX-6p/pORF5,表达并纯化质粒蛋白pORF5,并鉴定其免疫原性。方法用PCR法扩增pORF5基因,定向插入pGEX-6p原核表达载体构建原核表达重组体pGEX-6p/pORF5,重组体经酶切、PCR及测序鉴定后,经IPTG诱导在大肠杆菌中表达pORF5融合蛋白,融合蛋白纯化后免疫BALB/c鼠,ELISA及Western-blot分析pORF5质粒蛋白免疫原性及免疫反应性。结果PCR扩增出795 bp基因片段,序列测定证实重组质粒插入片段与GenBank登陆的D型Ct一致;pORF5融合蛋白在大肠杆菌中获得高效表达;ELISA检测免疫小鼠的血清效价为1∶25 600。结论pORF5蛋白在原核细胞中可有效表达并具有较好的免疫原性,为进一步研究该蛋白的结构功能、阐明Ct的致病机制、研制基因工程疫苗提供了有利的条件。

[ Objective ] To construct the recombinant plasmid containing pORF5 gene from Chlamydia trachomatis and study its immunogenicity in mice. [Methods] pORF5 gene was amplified from the genomie DNA of Ct serovar D by PCR and cloned into appropriate site of pGEX-6p vector to construct pGEX-6p/pORF5. After identification by PCR, restrictive enzymes digestion and sequencing, the recombinant plasmid was transformed into E.coli to express the fusion protein. The fusion protein was used to immunize BALB/c mice. ELISA and Western bolt were carried out to identify the immunogenicity and immunoreactivity. [Results] The pORF5 gene about 795bp was obtained. The DNA sequence of pORF5 gene was consistent with the published nucleotide sequence, pORF5 was suecessfully expressed as GST fusion proteins in E.coli. The ELISA result showed the specific antibody titer of pORF5 reached 1：25600. [Conclusion] pORF5 protein was successfully expressed in prokaryotic system and the specific anti-pORF5 antibody was induced in BALB/c mice, which lay a foundation for the further study on pORF5 protein function and vaccine development.