日本血吸虫Mf2基因的亚克隆表达与免疫保护效果研究

Subcloning,expression and vaccination studies with Schistosoma japonicum Mf2 gene

目的 在大肠杆菌中高效表达日本血吸虫 (Schistosomajaponicum Sj)Mf2蛋白 ,并对表达产物进行免疫保护效果测定。 方法 将SjMf2基因亚克隆至pCEX -5X -3原核表达载体 ,以GST融合蛋白的形式在ER2 688中表达 ,表达产物免疫小鼠 ,免疫用抗原剂量为 5 0 μg 次 鼠 ,对照组分别注射FCA或PBS ,在 0、2、6周共免疫 3次。第 3次免疫后 2周进行攻击感染 ,42d后杀鼠 ,观察减虫和减卵效果。 结果 在IPTG诱导下 ,表达载体中的SjGST基因与重组的SjMf2基因在大肠杆菌中得以高效表达 ,形成了SjGST -Mf2融合蛋白 ,用这种融合蛋白免疫小鼠 ,诱导产生了2 7 75 %的减虫率和 45 7%的每雌肝组织虫卵减少率 (LEPF)。 结论 SjMf2基因亚克隆至pGEX -5X -3载体后可在大肠杆菌中高效表达 ,表达产物能诱导小鼠产生一定程度的抗日本血吸虫保护性免疫力。

Objective To subclone and express of Schistosoma japonicum(Sj.)Mf2 gene in E.coil and to evaluate the immunoprotective effect of the recombinant molecules. Methods SjMf2 was expressed as a glutathione-S-transferase(GST)fusion protein by cloning into the prokaryotic expression vector pGEX-5X-3. The expressed proteins were vaccinated into mice for evaluation of the immunoprotective effect. The mice in vaccination groups were immunized subcutaneously with 50μg recombinant protein emulsified with Freund's complete adjuvant(FCA),and those in control groups were injected FCA or PBS at week 0,2,6. The mice were challenged by cercariae two weeks after the final injection,and were sacrificed 42 days after challenge. Worm burden and liver eggs per female(LEPF)were counted. Results Induced by IPTG, fusion protein SjGST-Mf2 were high expressed in E.coil in insoluble form, the inclusion bodies. The worm and the LEPF reduction rate were 27.75% and 45.57% respectively. Conclusion SjMf2 gene could be high expressed in E.coil after subcloned in pGEX-5X-3.Fusion protein Sj GST-Mf2 can significantly induce anti-infection immunity.