幽门螺杆菌cagA蛋白的克隆表达与鉴定

目的　构建表达幽门螺杆菌 (Hp)毒素相关基因A蛋白 (cagA)的重组质粒 ,并在大肠杆菌中表达获得基因重组蛋白 ,为检出幽门螺杆菌致病株和运用于临床检测Hp感染奠定基础。 方法　PCR扩增出编码毒素相关基因蛋白DNA片段后构建出重组质粒 pET -cagA ,并经质粒酶切筛选、DNA测序、IPTG诱导DE3 表达融合蛋白和Western blot予以证实。表达的融合cagA蛋白经过纯化和凝血酶酶切验证cagA蛋白抗原性。 结果　克隆的cagA核苷酸序列与GeneBank(AB116 74 4 )公布的序列相比较 ,同源性达 99.34% ,推定氨基酸序列同源性为 99.0 1%。SDS -PAGE和Western blot检测到带有His tag的约 4 0kD融合蛋白中表达。融合蛋白酶切后能与抗cagA人抗血清发生阳性反应。 结论　重组cagA的原核表达质粒构建正确 ,蛋白表达成功 ,具有反应原性

Expression and identification of cagA cloned from Helicobacter pylori

Objective To construct the recombinant plasmid of protein cagA (cytotoxin-associated gene A) of Helicobacter pylori and to express the fusion protein in E.coli DE_3, for the purpose of detection of wild pathogenic species and patients infected with Hp. Methods cagA gene was amplified by PCR and was cloned into prokaryotic expression plasmid pET-28a to construct a recombinant pET-cagA, which was verified by restriction nuclease, DNA sequencing. A recombinant protein was expressed in E.coli DE_3 induced with IPTG and its reactivity was tested by Western-blot test. After cut by thrombase to dismiss the his-tag, the purified protein was identified by Western-blot to test its antigenicity. Results DNA sequence analysis showed that the nucleic acid sequence of cagA gene was in 99.34% of homology and the putative sequence of amino acids was in 99.01%, compared with that (AB116744) published in GeneBank. A 40 kD fusion protein was detected by SDS-PAGE and its His-tag was recognized by its MAb from mouse in Western-blot. The serum antibody from human could recognize the recombinant protein or cagA. Conclusion The construction of plasmid recombinant encoding cagA of Helicobacter pyori is correct. Fusion protein cagA with antigenicity can be expressed in E.coli.