细粒棘球绦虫FABP与Eg95融合基因的表达与抗原性鉴定

目的构建细粒棘球绦虫脂肪酸结合蛋白(FABP)和Eg95两个保护性抗原基因的融合基因,并研究其重组蛋白的免疫学特性。方法以细粒棘球绦虫青海绵羊株保护基因FABP和Eg95的cDNA为模板,通过编码4个甘氨酸残基的连接序列,用非对称聚合酶链反应扩增融合基因FABP·Eg95,克隆至表达载体pET28a(+)中,在大肠埃希菌BL21(DB3)中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定。通过Ni-IDA亲和层析获得高纯度目的蛋白,利用蛋白质印迹(Western blotting)分析重组蛋白的免疫反应性。结果获得的融合基因FABP·Eg95长约795bp,双酶切和测序鉴定结果均显示pET-28a(+)-FABP-Eg95重组质粒构建成功。SDS-PAGE结果表明,重组质粒pET-28a(+)-FABP-Eg95在大肠埃希菌BL21(DB3)中获得高效表达,表达相对分子质量(Mr)约为31000的重组蛋白,主要以包涵体形式存在,经亲和层析获得目的蛋白。Western blotting分析结果显示,重组蛋白与细粒棘球蚴病患者血清有良好的免疫反应性,而不与健康人血清和血吸虫病患者血清反应。结论 FABP·Eg95融合基因构建成功,纯化的重组蛋白具有一定的抗原性。

Expression and characterization of the FABP and Eg95 fusion gene from Echinococcus granulosus

Objective To construct and express a fusion gene of fatty acid binding protein （FABP） with Eg95 which are protective antigen genes of Echinococcus granulosus, and investigate the immunological characteristics of the recombinant protein. Method Using cDNA fragments encoding FABP and Eg95 genes from E. granulosus Qinghai sheep strain as templates, a fusion gene FABP·Eg95 was amplified by asymmetric polymerase chain reaction through a linking sequence encoding four glycine residues. The PCR products of fusion gene were subcloned into the pET28a （+） vector. The recombinant plasmid was transformed into Escherichia coli BL21（DE3） cells and induced with IPTG. The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography, and its immunogenicity was analyzed by Western blotting. Results The fusion gene length was about 795 bp. Double enzyme digestion analysis and DNA sequencing confirmed that the fusioin gene was cloned into pET-28a （+）. The recombinant protein （M_r 31 000） was expressed in inclusion body in E. coli expression system, and was purified by Ni-IDA affinity chromatography. Western blotting analysis testified that the recombinant protein could be recognized by sera of cystic echinococcosis patients, but not by sera of healthy persons and schistosomiasis patients. Conclusion The FABP·Eg95 fusion gene has been constructed, and the purified recombinant protein has been confirmed with immunogenicity.