日本血吸虫M_r为8000钙结合蛋白基因的表达及其融合蛋白免疫反应性的评价

Expression of Schistosoma japonicum8kDa calcium-binding protein gene and evaluation of the immunoreactivity of its fusion protein

目的将亚克隆在pET32a(+)质粒上的日本血吸虫Mr为8 000钙结合蛋白(calcium-binding protein, CBP) 基因进行表达及蛋白纯化,并对表达产物用Western blotting 及ELISA方法进行免疫反应性的评价。方法将重组表达质粒pET32a(+)-CBP 转化入宿主菌BL21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;用金属Ni鏊合物亲和层析树脂(Ni-NTA)柱子纯化目标蛋白;将SDS-PAGE后的蛋白从凝胶转移到聚氟乙烯膜上,分别用6-组氨酸(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,ELISA法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Mr=29 000处有一表达条带,该蛋白与硫氧还蛋白融合表达,带有6-His基团, 用抗6-His抗体做免疫印迹有特异性的反应条带,而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都没有特异性的目标反应带出现,ELISA 的结果显示该蛋白与血吸虫感染兔血清没有免疫反应性。结论CBP蛋白与硫氧还蛋白融合表达后为可溶性蛋白且相对分子质量约为29 000,该蛋白与尾蚴感染兔血清及紫外照射减毒尾蚴免疫的兔血清均无免疫反应性。

Objective To obtain the expression of Schistosoma japonicum8 kDa calcium-binding protein gene subcloned into the pET32a(+) soluble expression plasmid and carry out purification and immunoreactivity assessment of the expressed pro-tein. Methods The recombinant plasmid was transferred to BL21 E.coli. strain, and induced with IPTG before bacterial lysis ultrasonically. The supernatant was analysed by SDS-PAGE after centrifugation. The target protein was purified with Ni-NTA agarose and transferred to the PVDF film for immunoreactivity test by Western blotting with anti-6-His antibody, the serum of rabbits 6 weeks after cercariae infection and the serum from UV-attenuated cercariae-immunized rabbits. The purified protein was used as the coating antigen for enzyme-linked immunosorbent assay (ELISA) to examine the sera from normal and cer-cariae-infected rabbits. Results The molecular weight of the target protein was 29 000 after IPTG induction. The fusion pro-tein presented a single band immunoreactive to anti-6-His antibody, which was not reactive to the serum from cercariae-infect-ed or UV-attenuated cercariae-immunized rabbit. Conclusions The calcium-binding protein is expressed as thioredoxinfusion protein with a relative molecular weight of about 29 000. The fusion protein possesses no immunoreactivity to cercariae-in-fected or UV-attenuated cercariae-immunized rabbit sera, possibly because of the specificity of the protein at the cercariae stage.