幽门螺杆菌粘附素基因hpaA的克隆、表达及鉴定

Cloning and Expression of Adhesion Gene HpA of Helicobacter pylori and Ident ification of Expressed Product

目的 克隆幽门螺杆菌粘附素基因hpaA ,构建其原核表达系统并鉴定融合蛋白免疫原性。方法 采用PCR技术从幽门螺杆菌总DNA中扩增hpaA基因 ,T -A克隆后测定核苷酸序列 ,构建pET30a的HpaA表达载体 ,在E .coliBL2 1DE3宿主菌中用IPTG诱导表达 ,Ni2 + 柱纯化后经Westernblot鉴定其免疫原性。结果 所克隆的hpaA基因与报道的相应核苷酸序列同源性为 94 . 8%～ 97. 3% ,氨基酸序列同源性为 94 . 6 %～ 97 .7% ,在 134～ 139位存在一段KRTIQK结构 ,HpaA融合蛋白在pET30a载体中可高效表达 ,经纯化后可获得高纯度的重组蛋白。结论 成功构建HpaA原核表达系统 ,所表达的融合蛋白具有较好的免疫原性 ,可作为Hp疫苗的候选抗原。

Objective To clone the adhesion gene HpaA of Helicobacter pylori (Hp),construct a prokaryotic expression system of the gene and i dentify the immunogenicity of expressed fusion protein.Methods Amplify HpaA gene from the total DNA of Hp by PCR and subject to T-A clonin g an d nucleotide sequencing.Insert the gene into plasmid pET30a,transform the constr ucted recombinant plasmid into E.coli BL21DE3 host bacterial strain and expr ess under indution of IPTG.Purify the expressed product by Ni 2+ chelating Sepharose chromatography and identify its immunogenicity by Western blot. Results The homologies of nucleotide and deduced amino acid sequence s of the cloned HpaA gene to those reported were 94 8%-97 3% and 94 6%-97 7% respectively.A KRTIQK fragment was observed at sites 134-139.HpaA fusion pro tein was highly expressed using pET30 vector,and a recombinant protein with high purity was obtained after purification. Conclusion A prokaryotic expression system for HpaA gene was successful ly constructed.The expressed fusion protein showed good immunogenicity and may b e used as a candidate antigen of Hp vaccine. [