重组日本血吸虫极低密度脂结合蛋白的抗原性及诊断价值的研究

Antigenicity and the immunodiagnostic potential of Schistosoma japonicum specific very low-density lipoprotein-binding protein (SVLBP) expressed in E.coli

目的 探讨重组日本血吸虫特异性极低密度脂结合蛋白 (SVLBP)的抗原性及其诊断价值。方法 SVLBP基因亚克隆入pQE - 30表达载体 ,克隆菌在IPTG诱导下表达重组蛋白 ;非变性条件下制备克隆菌裂解液 ,用亲和层析法纯化重组蛋白 ;用Westernblot鉴定其抗原性 ;纯化SVLBP重组蛋白免疫家兔获得抗血清 ;以SVLBP重组蛋白为抗原 ,ELISA法检测血吸虫感染者血清及健康人血清。结果 克隆菌在IPTG诱导下高表达SVLBP重组蛋白 ;该重组蛋白诱导家兔产生单特异抗体 ,抗体滴度为 1∶6 4 0 0 ;SVLBP重组蛋白与血吸虫病人血清有较强的反应 ;以该蛋白为抗原检测 36人份血吸虫感染者血清 ,阳性率为 83.3% ,5 0人份健康人血清 ,假阳性率为 2 % (1/5 0 ) ,10人份肺吸虫病人血清 ,1例阳性 ,交叉反应率为 10 % (1/10 )。结论 SVLBP重组蛋白有较好的抗原性 ,以该重组蛋白为抗原检测日本血吸虫病患者血清中特异性抗体 ,敏感性和特异性均较高 ,具有一定的诊断价值。

To investigate the antigenicity and the immunodiagnostic potential of recombinant SVLBP expressed in E.coli. the gene encoding SVLBP was subcloned into the expression vector,pEQ-30,and the clone pEQ-30-SVLBP was induced to express sufficient recombinant protein by IPTG.Lysate of cells was prepared with non-urea native buffers,then Ni-NTA Superflow was used to purify the recombinant protein from the supernant of lysate.Healthy rabbits were immunized with the purified recombinant SVLBP to evaluate its immunogenic property.Its antigenicity was analyzed with Western blot.And the diagnostic potential of recomb inant SVLBP was evaluated by ELISA.It was found that high expression of the recombinant SVLBP protein was induced with the introduction of IPTG.High-level monospecifc antibodies against SVLBP were induced in rabbits immunized with purified recombinant protein.The titer of antibodies was as high as 1∶6 400.The recombinant SVLBP predominantly reacted with IgG in sera from patients with Schistosomiasis.In IgG ELISA,recombiant SVLBP showed 83.3% sensitivity for patients with schistosomiasis and 98% specificity for healthy donors.One tenth of the sera from patients infected with Paragonimus westermni showed cross reaction with this protein.It concludes that the recombinant SVLBP is a potent diagnostic reagent for Schistosomiasis japonica .