幽门螺杆菌中性粒细胞激活蛋白的表达及免疫原性

目的克隆幽门螺杆菌(Hp)中性粒细胞激活蛋白(NAP)基因并制备其原核表达系统,研究其表达蛋白的免疫原性,为Hp疫苗研制提供理论依据。方法采用聚合酶链反应(PCR)扩增Hp-NAP基因,测序并作同源性分析后,亚克隆入原核表达载体pET-22b,转化表达菌BL21-CodonPlus(r)(DE3),IPTG诱导表达,SDS-PAGE分析,Western blot鉴定其免疫原性。结果PCR扩增一408 bp产物,与基因库中相关序列具有高度同源性(>98%)。重组质粒pET-22b-nap转化BL21-CodonPlus(r)(DE3)后表达一相对分子质量约19 000的融合蛋白,能与Hp全菌抗体产生结合反应,Western blot显示特异的单一条带。结论成功构建Hp-NAP原核表达系统,所表达NAP融合蛋白具有良好的免疫原性和免疫反应性,可作为Hp疫苗的候选抗原。

Expression and immunogenicity study of Helicobacter pylori neutrophil activating protein

Objective To clone the Helicobacter pylori(H.pylori) neutrophil activating protein(NAP) gene and construct its prokaryotic expression system, and to study the immunogenicity of the expressed fusion protein in order to provide a theoretical basic. Methods The Hp-NAP gene was cloned by PCR and subcloned into an prokaryotic expressioin vector pET-22b after sequencing and BLAST analysis.Then the identified recombinant plasmid pET-nap was transfected into E.coli BL21-CodonPlus(r)(DE3) and induced to express fusion protein by IPTG.SDS-PAGE and Western blot analysis followed for study of its immunogenicity. Results A 408 bp segment of Hp-NAP was cloned and subcloned into pET-22b vector successfully.BLAST analysis revealed that it had high homology with those corresponding sequences in GenBank(more than 98%).A fusion protein about 19 000 U was expressed by E.coli BL21-CodonPlus(r)(DE3) transfected with recombinant plasmid pET-nap and had a specific reaction with whole H.pylori cell antibody.No corresponding strap was found in the control. Conclusions An expression system with high efficiency of Hp-NAP has been constructed successfully.The expressed NAP protein with good immunogenicity and immunoreactivity can be used as a candidate antigen for H.pylori vaccine development.