肺炎衣原体蛋白激酶cpn0148的克隆表达和定位 - 生物谷.doc

肺炎衣原体蛋白激酶Cpn0148的克隆表达和定位 陈 凡1* 程 文2 (1. 中南大学湘雅医院心血管内科 湖南 长沙 410008)(2. 中南大学基础医学院免疫学系 湖南 长沙 410078) 摘 要: 目前认为肺炎衣原体(Chlamydia pneumonia, Cpn)除导致呼吸道疾病外, 也是与冠心病相关的重要病原体。作为一种细胞内寄生的病原菌, Cpn激活宿主细胞信号通路, 维护其在细胞内生长代谢, 并导致疾病。肺炎衣原体基因Cpn0148可编码真核细胞样的丝/苏氨酸蛋白激酶, 利用PCR技术扩增全长Cpn0148 ORF, 将其定向插入pGEX-6p原核表达载体, 在大肠杆菌XL-1blue中表达, 测序显示Cpn0148 ORF全长1860 bp, 编码619个氨基酸, 分子量大约70 kD, 将Cpn0148- GST融合蛋白免疫BALB/c小鼠获得抗Cpn0148多克隆抗体, 间接免疫荧光法检测显示Cpn0148表达在菌体上。Cpn0148蛋白质的获得为进一步研究Cpn与宿主细胞的相互作用奠定了基础。 关键词: 肺炎衣原体, Cpn0148, 丝/苏氨酸蛋白激酶 Cloning and Localization of the Ser/Thr Protein Kinase Cpn0148 from Chlamydia pneumoniae CHEN Fan1* CHENG Wen2 (1. Xiang-ya Hospital of Central South University, Hunan, Changsha 410008, China)(2. Basic Medical School of Central South University, Hunan, Changsha 410078, China) Abstract: Chlamydia pneumonia(Cpn) is thought to be one of the important pathogens which are associated with coronary heart disease. It can induce the activation of host signaling pathways to maintain the survival and metabolism of itself in the cell, and finally leads to disease. The ORFs of Cpn0148 encoding eukaryote-like serine/threonine protein kinase was amplified from Chlamydia pneumonia serovar AR39 genomic DNA. The amplified product was cloned in pGEX-6p and expressed in XL-1blue. DNA sequencing showed that the whole ORF is about 1860 bp, encoding a protein with 619 amino acid. The molecular weight of Cpn0148 is about 70 kD. Polyclonal antibody was obtained from immune BALB/c mice with the Cpn0148-GST fusion protein, and the expressing of Cpn0148 was detected on the bacteria organism by immunoflouresence. The finding of Cpn0148 protein is very helpful foundation for further study of the host-bacteria interaction. Keywords: Chlamydia pneumonia, Cpn0148, Serine/threonine protein kinase肺炎衣原体(Chlamydia pneumonia, Cpn)是一种细胞内寄生的革兰氏阴性菌, 可导致多种疾病, 目前研究显示Cpn不仅可导致衣原体性肺炎、支气管炎、哮喘等疾病, 也与冠状动脉粥样硬化有关[1,2], 日益受到人们的关注。 外界环境刺激真核细胞产生反应是通过信号转导来完成的, 蛋白磷酸化是信号转导主要机制, 信号转导的调节主要通过蛋白激酶和磷酸酶对丝氨酸、苏氨酸、酪氨酸残基磷酸化和去磷酸化而完