幽门螺杆菌cagA克隆表达

Cloning and expression of cagA gene of Helicobacter pylori

克隆表达 4株幽门螺杆菌的cagA基因 ,以方便地获得大量CagA蛋白和重组表达质粒 ,为临床诊断CagA阳性幽门螺杆菌感染 ,以及进一步研究不同类型CagA功能及其与疾病关系提供材料。PCR扩增幽门螺杆菌的cagA基因 ,克隆至PinPointTM Xa 1T载体 ,酶切鉴定连接方向 ,IPTG诱导正向连接克隆表达CagA融合蛋白并进行SDS PAGE和Westernblots鉴定。结果显示PCR扩增得到 3.5～ 3.8kb的cagA基因 ,PCR及酶切鉴定得到正向连接的重组克隆 ,SDS PAGE及Westernblots证实正向连接的重组克隆表达CagA融合蛋白。构建了 4种cagA的重组表达质粒 ,通过转化同一宿主菌可研究不同CagA的功能和致病性差异 ;通过亲和层析纯化融合蛋白可获取大量CagA蛋白 ,用于血清学诊断CagA阳性幽门螺杆菌感染 。

To clone and express the cagA genes of four different Helicobacter pylori strains, so as to provide materials for investigating the function of different CagA protein and their association with diseases, the cagA genes of four Helicobacter pylori strains were amplified by PCR, and cloned into pinpoint TM Xa-1 T vector to construct recombinant plasmids, of which generate in-frame recombinant protein were verified by PCR and digesting with restrict enzymes, SDS-PAGE and western blots of CagA fusion protein induced by IPTG. The results showed that PCR products of 3.5-3.9kb of cagA genes were obtained. the recombinant plasmids of cagA inframe were verified by PCR and digesing with restrictive enzymes, and revealed to express CagA fusion protein by SDS-PAGE and western blots.So the recombinant plasmids expressing CagA fusion proein have been constructed for determining CagA-positive H.pylori infection in serelogical method and further investigation of the function and pathogenicity of different CagA types.