幽门螺杆菌多亚单位融合蛋白疫苗的构建及免疫特性

目的构建幽门螺杆菌HspA、HpaA、UreB多亚单位融合蛋白疫苗,并检测其免疫原性及免疫保护性。方法用PCR从Hp基因组中分别扩增HspA、HpaA、UreB3个基因片段,重叠延伸PCR将其构建成融合基因hhu,并克隆至原核表达载体pET-28a(+)中,经酶切、测序验证后,转化E.coli BL21(DE3)中,IPTG诱导表达,AKATA纯化仪纯化。纯化后蛋白口服免疫BALB/c小鼠,ELISA检测小鼠特异性抗体,末次免疫后进行攻毒试验,检测其免疫保护性。结果酶切及测序证实已成功构建HspA、HpaA、UreB融合基因的表达载体phhu,SDS-PAGE及Western blot证实融合蛋白的表达,表达率为26·8%,纯化的融合蛋白纯度大于90%,口服免疫小鼠可产生特异性抗体,保护率达89·6%。结论所获得的融合蛋白保持了亚组分蛋白各自的免疫原性和免疫保护性,为幽门螺杆菌多亚单位疫苗的深入研究奠定了基础。

Construction and Immunological Property of Fusion Protein Vaccine with HpaA,HspA and UreB of Helicobacter pylori

Objective To construct the fusion protein vaccine with HpaA,HspA and UreB of Helicobacter pylori(Hp) and study its immunological property.Methods Amplify HspA,HpaA and UreB gene fragments from Hp genome by PCR and construct fusion gene hhu by SOE PCR.Clone the constructed fusion gene into prokaryotic expression vector pET-28( ),and transform the recombinant plasmid to E.coli BL21(DE3) for expression under induction of IPTG.Purify the expressed fusion protein by AKATA Explore 100.Immunize BALB/c mice with the purified fusion protein by oral route and determine the serum specific antibody by ELISA.Challenge the immunized mice with Hp and evaluate the immune protection.Results Restriction analysis and sequencing result proved that the expression vector phhu was successfully constructed.SDS-PAGE and Western blot showed that the expression level of fusion protein was 26.8%.The fusion protein reached a purity of more than 90% after purification and induced specific antibody in sera of immunized mice.The protective rate of the fusion protein against challenge with Hp was 89.6%.Conclusion The expressed fusion protein showed good immunogenicity and immune protection,which laid a foundation of further study on Hp subunit vaccine.