摘要
目的 将亚克隆在pET32a(+)质粒上的日本血吸虫蛋白酶体激活因子PA2 8亚单位基因进行表达及蛋白纯化 ,并对表达产物用Western -blot及ELISA方法进行免疫反应性的评价。方法 将重组表达质粒 pET32a(+) -PA2 8转化入大肠杆菌BL2 1中 ,IPTG诱导表达后超声裂菌 ,离心后取上清进行SDS -PAGE ,观察融合蛋白的表达情况 ;用Ni-NTA柱子纯化目标蛋白 ;将SDS -PAGE后的蛋白从凝胶转移到PVDF膜上 ,分别用 6 -His抗体、日本血吸虫尾蚴感染 6周的兔血清及紫外照射减毒尾蚴免疫的兔血清作一抗进行免疫印迹 ,将纯化后的融合蛋白作为包被抗原 ,检测正常兔血清及感染兔血清 ,对该蛋白的免疫反应性作出评价。结果 重组菌株用IPTG诱导后于 4 8kDa处有一表达条带 ,该蛋白与硫氧还蛋白融合表达 ,带有 6 -His,用抗 6 -His抗体做免疫印迹有特异性的单一反应带 ,用尾蚴感染兔血清及紫外照射减毒尾蚴免疫的兔血清做免疫印迹 ,结果显示该目的蛋白带与以上多抗具有敏感特异的反应条带。ELISA的结果显示该蛋白与血吸虫感染兔血清具有较敏感特异的免疫反应性。结论 PA2 8蛋白与硫氧还蛋白融合表达后为可溶性蛋白且分子量约为 4 8kDa ,该蛋白与血吸虫感染兔血清及紫外照射减毒尾蚴免疫的兔血清具有较敏感特异的免疫反应性。
To evaluate the expression,identification,purification and immune reactivity of the proteasome activator PA28,subunit whose cDNA sequence had been subcloned in to the pET32a(+) soluble expression plasmid previously,the recombinant plasmid was transformed into E.coli BL21 and was induced with IPTG.The lysed bacteria was obtained by ultrasonic treatment,and the supernatant was analyzed with SDS-PAGE after centrifugation.The target protein was purified with Ni-NTA column agarose,and the proteins on the gel were transferred to PVDF film.Then, the immune reactivity was tested by Western blotting by using the anti-6-His antibody,6 weeks immune sera of rabbits infected with cercariae and the virradiated,attenuated cercariae infected rabbit sera.The purified protein was used as the coating antigen in ELISA testing to test the cercariae infected rabbit sera and normal rabbit sera.It was found the molecular weight of the target protein was 48 kDa after being induced with IPTG,and the fused protein had a single band of positive immune ractivity with anti-6-His antibody and had a positive result with the cercariae infected rabbit sera and attenuated cercariae infected rabbit sera.It concludes that the PA28 protein is a soluble protein with molecular weight of 48kDa after the fusion expression with thioredoxin.This protein shows a positive immune reactivity with the cercariae infected and UV attenuated cercariae infected rabbit serum.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2004年第3期183-185,189,共4页
Chinese Journal of Zoonoses
基金
联合国发展开发署 /世界卫生组织热带病研究和培训特别规划署资助 (IDNo .
A0 0 690A0 0 191)
