幽门螺杆菌临床菌株2148bp的cagA基因片段克隆、表达及免疫性鉴定

Cloning and expression of fragment with 2148 bp from cagA gene from an isolate of Helicobacter pylori and identification of immunogenicity of the recombinant protein

目的 克隆幽门螺杆菌 (Helicobacterpylori,Hp)临床菌株的cagA基因片段 ,构建原核表达系统 ,鉴定重组表达产物的免疫原性。方法 由于cagA基因核苷酸序列变异较大 ,选择序列相对稳定的 2 14 8bp为目的片段 (cagA1)。采用高保真PCR扩增幽门螺杆菌临床分离菌株Y0 6中的目的片段cagA1,T -A克隆后测序。构建 pET32a的cagA1片段表达载体 ,在E coliBL2 1DE3宿主菌中用不同浓度的IPTG诱导表达。采用Ni-NTA亲和层析法收集表达的目的重组蛋白 (rCagA1)。采用Hp全菌抗体的Westernblot鉴定rCagA1免疫反应性 ,免疫双扩散试验鉴定rCagA1免疫家兔的抗血清效价。 结果 PCR获得预期大小的目的扩增条带。与文献报道比较 ,克隆的cagA1核苷酸和氨基酸序列同源性分别为 94 83%和93 30 %。所构建的原核表达系统 pET32a -cagA1-E coliBL2 1DE3目的重组蛋白 (rCagA1)表达量为细菌总蛋白的 30 %左右。Westernblot证实rCagA1能与Hp全菌抗体发生特异性免疫结合反应。兔抗rCagA1血清的免疫双扩散效价为 1∶4。结论 本文成功地构建了HpcagA基因片段cagA1的高效原核表达系统 ,所表达的rCagA1具有良好的免疫原性和免疫反应性 ,为进一步研制cagA及其抗体检测试剂盒奠定了基础。

Because of high mutation of H.pylori cagA gene,a 2148bp fragment (cagA1) with relative conservation in the nucleotide sequence of the gene was selected to construct an expression recombinant vector for obtaining antigen used in detection kit.cagA1 fragment from clinical H.pylori strain Y06 DNA was amplified and then a prokaryotic expression system pET32a-cagA1-E.coli BL21DE3 was constructed.SDS-PAGE,western blotting and immunodiffusion assay were applied to determine the expression,immunoreactivity and antigenicity of the target recombinant protein (rCagA1),In comparison with the reported sequences,homologes of nucleotide and putative amino acid sequences of the cloned cagA1 fragment were 94.83% and 93.30%,respectively.The amount of rCagA1 expression produced by the expression system pET32a-cagA1-E.coli BL21DE3 was approximate 30% of the total bacterial proteins.The rCagA1 was able to combine with the commercial antibody against whole cell of H.pylori and could induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1∶4.Therefore,we successfully constructed a prokaryotic expression system of H.pylori cagA1 fragment and the expressed rCagA1 showed qualified immunogenicity and immunoreactivity,that establishing a favorable foundation for further development of CagA and CagA antibody detection kit.