肺炎衣原体CPn0308的基因克隆及其内源性蛋白定位的研究

目的克隆肺炎衣原体基因CPn0308,表达融合蛋白,并制备抗体对其表达的内源性蛋白进行初步定位。方法根据STD基因库提供的信息设计引物,聚合酶链反应(PCR)克隆目的基因,用BamHI/NotI对克隆的目的基因和pGEX-6P2载体进行酶切,T4连接酶连接后,重组质粒经42℃转化XL1-blue细菌;用PCR进行初步筛选,交叉PCR对提取质粒进一步确定,最后对插入片断进行序列分析;用IPTG诱导阳性克隆的细菌使其表达GST融合蛋白,纯化后免疫小鼠制备抗体,使用IFA对内源性蛋白进行定位分析。结果克隆出肺炎衣原体基因CPn0308,全长为366bp,编码121个氨基酸;并表达了融合蛋白GST-CPn0308,分子量约为39kD;制备了抗体,IFA实验发现该蛋白初步定位于肺炎衣原体包涵体膜蛋白上。结论成功克隆肺炎衣原体基因CPn0308,其内源性蛋白初步定位于肺炎衣原体包涵体膜上。

Study on gene cloning of Chlamydial pneumonia CPn0308 and its endogenous localization

OBJECTIVE: To clone CPn0308 gene from Clamyida pneumonia and express its fusion protein, to make antibodies to fusion protein GST-CPn0308, and to further localize endogenous protein preliminarily using antibodies raised with CPn0308 fusion protein. METHODS: The open reading frame (ORF) coding for CPn0308 in the Chlamydia pneumonia AR 39 genome was cloned into the pGEX6p2 vector after it was cloned using PCR and digested by the restriction enzymes BamHI and NotI. The recombinant plasmid pGEX6p2-CPn0308 was transformed into XL1-blue bacteria and the gene CPn0308 was expressed as fusion proteins with the glutathione-s-transferase (GST) tagged to the N-terminus. The GST-CPn0308 fusion protein was used to immunize mice and the mouse anti-fusion protein antibody was used to localize the endogenous CPn0308 protein in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). RESULTS: The CPn0308 gene, which was 366bp in length,was successfully cloned and the GST fusion protein with molecular weight of 39kD was expressed. It was found that the hypothetical protein CPn0308 was located in the inclusion membrane of Chlamydia pneumonia-infected cells using IFA of mouse anti-fusion protein antibodies. CONCLUSIONS: Using antibodies raised with GST-CPn0308 fusion protein, the hypothetical protein CPn0308 was identified to be a Chlamydia pneumoniae inclusion membrane protein. It could be the potentially important role of inclusion membrane proteins in chlamydial interactions with host cells.