幽门螺杆菌粘附素基因hpaA的克隆、表达及鉴定

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。

Cloning and Expression of Adhesion Gene HpA of Helicobacter pylori and Identification of Expressed Product

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. [