幽门螺杆菌Ⅳ型分泌系统Cag3基因的克隆表达及其质谱鉴定

Cloning expression and identification by mass spectra of Cag3 gene of Helicobacter pylori type Ⅳ secretion system

目的构建表达幽门螺杆菌(Hp)Cag致病岛(Cag-PAI)编码的Cag3(hp0522)基因重组质粒,转入大肠杆菌中表达,纯化重组蛋白。方法以Hp 11637基因组为模板,应用PCR技术扩增Cag3基因片段,克隆至pGEM-T载体中测序,然后克隆到高效表达载体pET-28a(+)上,得到了重组表达载体pET-28a(+)-Cag3,转化表达宿主菌大肠埃希菌BL21;经IPTG诱导表达后,将大量表达的重组蛋白用Ni 2+-NTA亲和层析柱纯化;采用串联飞行时间质谱技术对Cag3-His融合蛋白进行鉴定。结果成功克隆了Cag3基因。经SDS-PAGE分析,表达蛋白相对分子质量与预期大小一致;质谱检测到Cag3重组蛋白的表达。结论成功构建了Cag3基因的原核表达载体pET-28a(+)-Cag3。

Objective To construct the recombinant plasmid of Cag3（HP0522） of Helicobacter pylori（Hp） NCTC 11637 of Ⅳ type secretion system and to express fusion protein in E.coli BL21.Methods The Cag3 gene was amplified by PCR from genome of Hp genome and inserted into cloning vector pGEM-T.After sequencing,the gene was cloned to the expression vector pET-28 to construct recombinant expression plasmids pET-28a（＋）-Cag3.Recombinant pET-28a（＋）-Cag3 was expressed in E.coli BL21.IPTG was used to induce expression,after which the recombinant protein was purified by Ni2＋-NTA column.Tandem time-of-flight mass spectrometry was applied to identify Cag3-His protein.Results The Cag3 was cloned successfully.SDS-PAGE analysis showed that molecular weight（MW） of the protein corresponded to the MW anticipated.Tandem time-of-flight mass spectrometry detected Cag3 fusion protein.Conclusion The recombinant expression plasmids pET-28a（＋）-Cag3 has been constructed successfully.