细粒棘球蚴铁蛋白基因的克隆重组高效表达及免疫学初步研究

目的构建细粒棘球蚴（Echinococcus granulosusEg）铁蛋白（ferritin）基因的原核表达重组质粒并表达、纯化该重组蛋白，初步研究其免疫反应。方法将细粒棘球蚴铁蛋白基因亚克隆于表达载体pGEX-6p-1，转化重组体到大肠杆菌B121中，在异丙基-β-D-硫代半乳糖苷（IPTG）诱导下表达；用SD争PAGE和Western—blot对表达产物进行初步鉴定，用切胶纯化的融合蛋白免疫BALB／c小鼠，通过Western-blot对该蛋白的免疫学特性进行初步研究。结果重组铁蛋白与GST以融合表达的形式在细菌中高效表达，表达产物为不可溶性的包涵体，蛋白分子量约为42kD。融合蛋白Egferritin／GST能被细粒棘球蚴免疫的家兔血清和特异性小鼠抗血清所识别，同时特异性小鼠抗血清可识别天然抗原囊液、原头蚴可溶性蛋白中约19kD的蛋白条带。结论成功地表达细粒棘球蚴重组铁蛋白，并且该蛋白有一定的免疫原性及抗原性。

High level expression and identification of recombinant ferritin of Echinococcus groanolosus

To construct the prokaryotic expression recombinant plasmid containing Echinococcus granulosus ferritin gene and to express, purify and study the immune responses of the recombinant gene induced product, this ferritin gene was subcloned into high-level expression vector pGEX-6p-1 and this recombinant expression vector was then used to transfer to E. coli BL21 cell. The recombinant fusion protein expressed in E. coli cell was induced with IPTG and identified by SDS-PAGE and Western blot. Meanwhile, the fusion protein purified with cutting gel was used to immunize BALB/c mice to investigate its immunological characteristics by means of Western blot analysis. The experimental results showed that the recombinant ferritin/GST fusion protein was highly expressed in E. coli BL21 cell and the expressed product was an insoluble protein with molecular weight of 42 kDa. This protein could be recognized by sera of rabbits immunized with E. granulosus and the specific antisera of mice immunized with the recombinant ferritin/GST fusion protein. This specific mouse antiserum could also recognize the 19 kDa band in native antigen cyst fluid and protoscoles of E. granulosus. It is concluded that the recombinant ferritin/ GST fusion protein is successfully expressed and purified with higher immunogenicity, thus it may be the suitable candidate for vaccine development.