幽门螺杆菌尿素酶表位疫苗的构建、表达及鉴定

Construction and Prokaryotic Expression of Fusion Protein Epitopes of Urease B Subunit of Helicobactor pylori

目的构建和表达幽门螺杆菌(Helicobacter pylori,Hp)尿素酶B亚单位(UreB)表位串联体(Uepi)与大肠杆菌不耐热肠毒素B亚单位(LTB)融合蛋白表位疫苗,并对其生物学及免疫学特性进行鉴定。方法设计引物,采用PCR法分别扩增UreB表位多肽编码基因Uepi和LTB编码基因,重叠延伸PCR法将两段基因拼接,T-A克隆后,构建融合基因表达质粒pET-22b(+)-Uepi-LTB,经酶切鉴定后转化E.coliBL21(DE3),IPTG诱导表达,并对表达产物进行鉴定。结果PCR扩增出206bp和336bp的目的片段,重叠延伸PCR扩增出524bp的融合目的基因片段。原核表达质粒pET-22b(+)-Uepi-LTB经酶切及测序鉴定,与设计序列一致。重组工程菌pET-22b(+)-Uepi-LTB/BL21经IPTG诱导,目的蛋白表达率约25%,SDS-PAGE分析相对分子质量约20000,目的蛋白以包涵体形式表达,纯化后蛋白纯度达96%,Westernblot鉴定该融合蛋白与兔抗LTB多抗血清可发生特异性结合。结论HpUreB表位串联体与LTB融合蛋白的表位疫苗经基因克隆,可获得高效表达,并显示出较好的免疫活性,为新一代Hp疫苗的研制奠定基础。

Objective To construct and express the fusion protein epitopes of Helicobactor pylori （Hp） urease B subunit （UreB） concatemer and E. coli heat-labile toxin B subunit（ LTB）, and analyze the biological and immunological characteristics of expressed product. Methods Amplify the genes encoding UreB epitope polypeptide and LTB by PCR respectively and link by overlap extension PCR. Aider T-A cloning,the linked gene Uepi-LTB was cloned into prokaryotic expression vector pET-22b （ ＋ ）, and the con- strueted recombinant plasmid pET-22b（ ＋ ）-Uepi-LTB was transformed to E. coli BL21 （DE3） for expression under induction of IPTG. Identify the expressed product by Tricine-SDS-PAGE and Western blot, and purify by SP Sepharose XL and Butyl Sepharose FF chroma- tography. Results Two gene fragments at the lengths of 206 bp and 336 bp respectively were amplified by PCR. However,by overlap extension PCR,a fusion gene fragment at the length of 524 bp was amplified. Both the results of restriction analysis and DNA sequencing of prokaryotic expression vector pET-22b （ ＋ ）-Uepi-LTB were consistent with those designed. The expressed product, with a relative molecular weight of about 20 000 ,existed in a form of inclusion body and contained about 25% of total somatic protein. Aider purification,the expressed protein reached a purity of 96%. Western blot showed specific reaction of expressed fusion protein with rabbit anti-LTB serum. Condutdon Fusion protein Uepi-LTB was successfully expressed in prokaryotic cells. The expressed product showed good immunogenicity, which laid a foundation of developing novel Hp vaccine.