沙眼衣原体隐匿质粒编码蛋白的表达和相互作用的-第三军医大学学报.docVIP

沙眼衣原体隐匿质粒编码蛋白的表达和相互作用的分析 高磊琼12，2，华子瑜12，沈犁2（1重庆医科大学附属儿童医院新生儿科，重庆 400014；2重庆医科大学附属儿童医院，儿童发育疾病研究教育部重点实验室、儿科学重庆市重点实验室、重庆市（儿童发育重大疾病诊治与预防）国际科技合作基地，重庆 400014）pgp1-4及pgp8分别克隆到pRAC和pRBR上，通过转录激活细菌双杂交系统分析蛋白相互作用。结果 Western blot结果显示Pgp1、Pgp3与大肠杆菌RpoA 氨基末端功能区（α-NTD）融合蛋白及Pgp1、Pgp3、Pgp4 与DNA结合蛋白λCI融合蛋白，能在大肠杆菌中表达。在报告菌株中，共表达与α-NTD融合的Pgp3蛋白（α- Pgp3）及与λCI融合的Pgp3蛋白（CI-Pgp3），导致报告基因β-半乳糖苷酶的活性升高pgp3编码的Pgp3能与自身相互作用，而其它蛋白间没有相互作用。 [关键词]：沙眼衣原体; 隐匿质粒; Pgp3; 蛋白相互作用 Protein-protein interaction analysis：a cryptic plasmid-encoded protein in Chlamydia trachomatis GAO Leiqiong1,2，Zhang Yan2, HUA Ziyu1,2, SHEN Li2 (1Neonatology Department, Chongqing Chlidren’s Hospital,Chongqing Medical Univercity,) (1. Department of Neonatology, Chongqing Children’s hospital, Chongqing medical university , Chongqing 400014, China; 2Ministry of education key laboratory of child development and disorders，key laboratory of Pediatrics committee of science and technology in Chongqing，Chongqing international science and technology cooperation center for child development and disorders，Chongqing Children’s hospital, Chongqing medical university, Chongqing 400014,China) ［Abstract］ Objective To analyze the interactions among proteins encoded by the plasmid in Chlamydia trachomatis. Method The DNA fragments of C. trachomatis plasmid genes, pgp1-4 and pgp8, were cloned into the vectors (pRAC and pRAC) and the target genes were in-frame fused toα-NTD and λCI, respectively. The expression of cloned genes in?Escherichia coli（E.coli） were determined by Western blotting. The protein-protein interactions were analyzed by a transcription-based bacterial two-hybrid system. Resultes The fusion proteins ofα-Pgp1,α-Pgp3,CI-Pgp1,CI-Pgp3 and CI-Pgp4 were expressed in E.coli by the addition of Isopropyl β-D-1-thiogalactopyranoside （IPTG）. In the presence of Pgp3 (encoded by plasmid pBRα-Pgp3 ), the expression of CI-Pgp3 activated transcription from a reporter gene in the bacterial two-hybrid assay strain. We did not detect interactions of