猪瘟病毒E2蛋白重复多表位基因的融合表达及其兔体免疫保护研究

目的：研究猪瘟病毒(CSFV)E2蛋白重复多抗原表位基因的融合表达及其免疫攻毒保护作用。方法：应用PCR方法扩增猪瘟病毒E2蛋白重复多抗原表位基因，构建重复多表位基因的原核重组表达质粒PGEX-3E，进行融合蛋白的表达和纯化。ELISA和Western blot方法测定单表位融合蛋白GST-E和多表位融合蛋白GST-3E与猪抗CSFV的阳性血清和兔抗E2阳性血清的反应性，并进行融合蛋白的兔体免疫及免疫攻毒保护的比较研究。结果：分子克隆和构建了原核重组表达质粒pGEX-3E，表达和纯化了融合蛋白GST-3E；单表位融合蛋白与重复多表位融合蛋白均能够与猪抗CSFV的阳性血清反应和兔抗E2的阳性血清产生免疫反应。在刺激兔体产生抗体方面，单表位融合蛋白刺激兔体产生抗体的能力较弱，而多表位融合蛋白则能够使兔体产生高效价的抗体。接种100MID50剂量猪瘟兔化弱毒(HCLV)进行的兔体免疫攻毒保护试验表明，空白对照组和载体蛋白GST免疫组无保护作用，单表位融合蛋白免疫组具有一定的免疫保护作用，而重复多表位融合蛋白免疫组则完全能够抵抗猪瘟病毒的攻击。结论：猪瘟病毒E2蛋白重复多表位的融合表达具有免疫保护作用，本实验的成功完成为猪瘟病毒多表位抗原的串联及多表位疫苗的研究奠定了基础。

Fusion expression of multiepitope gene of CSFV envelope glycoprotein E2 and its protection efficiency against homologous challenge in rabbits

Objective:To investigate the fusion expression of multiepitope gene of CSFV envelope glycoprotein E2 and its protection efficiency against homologous challenge in rabbits.Methods:The multiepitope gene of CSFV envelope glycoprotein E2 was amplified by PCR technique.The PCR product was cloned into pGEX-3X vector,then transformed into E.coli BL21 cells and expressed.The single epitope fusion protein (GST-E) and multieptope fusion protein (GST-3E) were analyzed by ELISA and Western blot with pig serum against CSFV and rabbit serum against E2.The protection efficiency of the GST-E and GST-3E was determined by challenge of hog cholera lapinized virus (HCLV).Results:The GST-E and GST-3E can interacted with pig serum against CSFV and rabbit serum against E2.The capacity of inducing antibody of GST-E by immunizing rabbit is low,but GST-3E is high.The rabbits challenged with 100 MID_(50) HCLV demonstrated that GST-E immunized rabbits showed a mild increase of body temperature,GST-3E immunized rabbits no body temperature increasing on the other hand the GST immunized and control rabbits body temperature increased and then recover normally.Conclusion:The GST-3E can induce an efficient immune protection against HCLV infection,and might provide a new kind of vaccine for CSFV.