【作者】 罗庆礼；

【导师】 沈继龙； 汪学龙；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2006， 硕士

【摘要】 目的：从本实验室保种的重组质粒pBK-CMV-SjiGST中扩增出日本血吸虫(中国大陆株)26 kDa谷胱甘肽-S-转移酶(SjGST)编码基因，克隆至表达载体pET28a(+)；分别诱导表达本实验室保种的含有日本血吸虫信号蛋白14-3.3(Sj14-3-3)编码基因的重组表达载体pET28a(+)-Sj14-3-3和构建的重组表达载体pET28a(+)-sjGST；制备并分别纯化出重组蛋白Sj14-3-3和SjGST；将两者联合应用，建立重组抗原间接ELISA方法(rSj-ELISA)诊断日本血吸虫病。棋盘滴定法确定最适抗原包被量和血清稀释度，用于急、慢性血吸虫病诊断，并与成虫粗抗原(sjAWA)的间接ELISA，以及间接血凝试验(IHA)和环卵沉淀试验(COPT)平行检测，比较几种方法的敏感性和特异性之间的差异。同时比较3种方法在华支睾吸虫和钩虫感染者之间的交叉反应性，以探讨重组抗原用于诊断血吸虫病的实用性和可靠性。 方法：提取含SjGST编码基因的重组质粒pBK-CMV-SjGST，PCR扩增出目的片断，将扩增产物克隆入表达载体pET28a(+)，诱导表达出rSjGST；同时诱导含Sj14-3-3编码基因的重组质粒pET28a(+)-Sj14-3-3表达出rSj14-3-3。SDS-PAGE和Western blotting验证表达量和免疫活性，并分别用亲和层析法纯化重组蛋白，并用Bradtbrd法检测二者的含量。将rSj14-3-3和rSjGST按1：1比例联合应用包被反应更多还原

【Abstract】 Objective: To approach the poteintial of the recombinant signaling protein 14-3-3 （rSjl4-3-3）and 26kD glutathione-S-transferase（GST） of Schistosoma japonicum in the diagnosis of schistosomiasis. Methods: The SjGST gene was amplified from recombinant plasmid pBK-CMV-SjGST by Polymerase chain reaction（PCR） and subsequently cloned into expression vector pET28a（+）. The rSj14-3-3 and rSjGST were expressed in E. coli and purified through the collumn of affinity chromatography with Ni²⁺（nickel sulfate, NiSO₄）. The rSj14-3-3 and rSjGST antigens were subjected to the serological diagnosis in the patients with acute and chronic schistosomiasis japonica using indirect ELISA. The sensitivity and specificity of the recombinant antigens used in ELISA （rSj-ELISA） were compared in parallel with the traditional methods of SEA-IHA and COPT. In addition to the sera of patients with acute and chronic schistosomiasis, the detected samples include the sera of individuals with clonorchiasis and hookworm infections and those from the areas with and without schistosomiasis. A paralled tests were also performed using the adult worm antigens（ SjAWAs） in ELISA. Results: The size of PCR products of SjGST was approximately 670 bp, which is更多还原