汉赛巴尔通体p7基因原核表达及间接elisa检测方法的建立.pdfVIP

摘 要 目的：为临床上应用间接ELISA 方法检测汉赛巴尔通体病原体，本文通过对汉 赛巴尔通体 P17 基因原核诱导表达，用获得的重组蛋白分析研究间接 ELISA 检测 方法的最佳条件。 方法：根据 Genebank 上的基因序列，对汉赛巴尔通体特有 P17 （片段长度为 525bp ）进行PCR 扩增，并克隆到pET-28a 载体上。将重组质粒转化到大肠杆菌DH-5α 中，用l mmol/LIPTG 诱导P17 蛋白表达，然后将Ni-NTA 柱纯化的目的蛋白作为抗 原建立汉赛巴尔通体的人临床间接ELISA 检测方法。 结果：成功获得了分子量为22.6KD 、浓度为3.44mg/mL P17 基因重组蛋白。 建立了间接ELISA 方法的最佳条件为：P17 基因蛋白稀释浓度为1.25µg/ml， 包被 37℃2 小时，5%脱脂奶37℃封闭1h，待检血清稀释度为1:400，37℃孵育1h，HRP- 羊抗人IgG 浓度 1:5000，37℃孵育 1h，底物37℃避光显色25min ，阴阳临界值为 0.1238 。利用已建立的ELISA 方法检测上海地区91 份和辽宁省393 份临床血清样 品，阳性血清分别为阳性率为24.17%和19.59%。 结论：成功建立了人临床汉赛巴尔通体抗体检测的间接ELISA 方法，该方法特 异性、重复性及敏感性高。此外，应用原核重组表达并纯化的抗原蛋白的方法，具 有成本低，操作简便，结果可信等特点，为将本研究中建立的ELISA 应用于生产的 实践和临床诊断中提供可靠的实验依据和理论基础。 关键词：汉赛巴尔通体；猫抓病；人畜共患病；原核表达；ELISA Prokaryotic Expression of P17 Gene of Bartonella henselae and Establishment of an Indirect ELISA for Detection of Expression Product Abstract Purpose: For check out the pathogen of Bartonella henselae by indirect ELISA method in clinically, established the optimum condition of indirect ELISA method using the recombination protein which was inducted expression of Bartonella henselae P17 gene. Methods: According to the gene sequences in Genebank, amplified the P17 by PCR, which is the unique gene of B. henselae (the size as 525bp), and cloned the gene to the vector pET-28a. Then transformed the recombinant expression plasmid into the E . coli DH-5α and induced the P17 protein expression by IPTG with a finial concentration 1mmol/L, and using the purified interest protein with the assay of Ni-NTA column as antigen established the Bartonella henselae indirect Elisa method in human clinically. Results: Successfully obtained, the Molecular Weight was 22.6KD