幽门螺杆菌双价亚单位分子内佐剂疫苗的构建表达与纯化

Expression and purification of fusion protein of Helicobacter pylori ahpC-ureB414 and E.coli heat-labile enterotoxin B subunit

目的克隆表达幽门螺杆菌外膜蛋白烷基过氧化氢还原酶ahpC、ureB414功能片段基因,与大肠杆菌不耐热肠毒素B亚单位构建融合基因ahpC-ureB414-ltB(aul)。方法采用重叠延伸PCR的方法,以AhpC做为融合蛋白前端,与UreB414功能片段及黏膜佐剂ltB依次串联,在片段间引入5个氨基酸的柔性Linker GGGGS进行连接;将融合基因构建在原核表达载体pET-22b(+)中,经酶切及测序鉴定后转化宿主菌E.coli BL21(DE3),IPTG诱导表达融合蛋白ahpC-UreB414-ltB(AUL)。融合蛋白经AKTA-explore纯化仪纯化融合蛋白。结果PCR扩增出1350bp的目的基因片段aul,工程菌pET22b-aul/BL21经IPTG诱导后,SDS-PAGE显示有新生的蛋白表达条带,相对分子质量约为50×103,与预期值一致,目的蛋白约占菌体总蛋白的28.7%,重组蛋白用Ni2+-NTA树脂提纯。纯化后的蛋白质经SDS-PAGE分析可见单一条带,图像软件分析表明纯度可达85%以上。Western blot显示重组蛋白质具有良好的免疫反应性。结论成功构建、表达融合蛋白rAUL,纯化后的融合蛋白保持各亚单位组分及佐剂的独立免疫反应性。

Objective To construct the fusion gene of H. pylori outer membrane protein AhpC, UreB414 and E. coli heat-labile enterotoxin B subunit. Methods We constructed the fusion gene aul including ahpC, ureB414 and hB gene by SOE PCR （splicing by overlap extension） , setting AhpC gene before UreB414-hB fusion gene and introducing a fixible linker GGGGS between them. Then the aul gene was constructed into the expression plasmid pET-22b（ ＋ ）. The fusion protein was expressed in E. coli BL21 induced by IPTG, confirmed by SDS-PAGE and Western blotting. The inclusion body was purified with AKTA-explore 100 system after washing. Results The fusion gene was successfully cloned into pET-22b（ ＋ ）. The gene segment inserted into the recombinant vector was identified by restriction enzyme digestion and sequencing, and proven to be con- sistent with that in GenBank. The relative molecular weight of the expressed product was 50 × 10^3. The purity of rAUL was up to 85% after Chelating Sepharose affinity chromatography. Conclusion The fusion proteins rAUL and rLUL were constructed and expressed successfully. The fusion proteins retain the immunoreaetivity of each of their components as well as the binding activity with GMI ganglioside.