重组日本血吸虫亲环素A的原核表达及免疫保护作用的研究

Prokaryotic expression and protective effect evaluation of recombinant Schistosoma japonicum CyPA

目的克隆、表达日本血吸虫亲环素A(Sj Cy PA)基因,并对重组蛋白的免疫保护效果进行实验室评价。方法构建p ET28-Sj Cy PA重组质粒,转化感受态大肠杆菌BL21/DE3。用IPTG诱导表达,经亲和层析纯化目的蛋白。用PBS、r Sj Cy PA分别免疫小鼠后进行日本血吸虫尾蚴攻击感染,观察其免疫保护效果。结果成功表达了可溶性Sj Cy PA蛋白并得到纯化,经Western blot鉴定为血吸虫蛋白。r Sj Cy PA免疫小鼠产生60.10%的减虫率和43.42%的减卵率,HE染色及Masson染色揭示实验组肝脏虫卵肉芽肿及纤维化明显减轻。结论 r Sj Cy PA具有较好的抗血吸虫感染和抗血吸虫病的作用,是一个有前景的血吸虫疫苗候选分子。

Objective To clone and express Schistosoma japonicum cyclophilin A in E.coli and evaluate its immunoprotective effect.Methods The recombinant plasmid p ET28a（＋）-Sj Cy PA was constructed and transformed into E.coli BL21.After induced by isopropyl-B-D-thiogalaetoside（IPTG）,the expressed target protein was purified by affinity-chromatography,and then verified by SDS-polyacrylamide gel electrophoresis（SDS-PAGE） and Western blotting.20 KM mice were randomly divided into 2 groups（n = 10）,which including PBS control group and r Sj Cy PA immunization group.All mice were immunized with PBS or r Sj Cy PA for three times with 2-weeks intervals.Six weeks later,all mice were sacrificed;the adult worms were collected and the worm reduction rate was calculated.Meanwhile,the liver was removed,sectioned and stained to count the eggs per mm2 to obtain the egg reduction rate.HE and Masson staining method was used to detect the hepatic granuloma and pathological changes.Results Soluble recombinant r Sj Cy PA protein was highly expressed in E.coli.The worm reduction rate in r Sj Cy PA vaccination group was 60.10%;the liver egg reduction rate was 43.42%.Egg granuloma and degree of liver fibrosis were significantly ameliorated.Conclusion r Sj Cy PA has relatively good protective effect against Schistosoma japonicum infection and schistosomiasis,and is a promising vaccine candidate for S.japonicum.