[幽门螺杆菌napA基因的克隆、表达及免疫反应]

[Clone, expression and immunoreaction of napA gene of Helicobacter pylori].

作者信息

Luo Jun, Long Bei-guo, Li Yan, Lai Jian-ping, Hu Ping, Zhang Wen-bing, Long Min

机构信息

Faculty of Microbiology, School of Public Health and Tropic Medicine, Southern Medical University, Guangzhou 510515, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 May;23(5):395-8.

PMID:17488595

Abstract

AIM

To construct a prokaryotic expression system of Helicobacter pylori(Hp) (neutrophil-activating protein) napA gene, analyze nucleic acid sequence and study its immunity and inflammation.

METHODS

napA fragment was amplified from Hp NCTC11639 chromosomal DNA by PCR. Its T-A was cloned, sequenced and compared with other Hp strains on the GenBank. Then the gene was cloned into pGEX-4T-1 fusion expression vector, expressed in E.coli and purified by GST-affinity chromatography. The purified product was used to screen 29 stains of mouse anti Hp monoclonal antibodies(mAb) and its immunity and inflammation analyzed with sera of Hp-infected patients by Western blot.

RESULTS

napA fragment was composed of 435 base pairs (GenBank No.DQ341279) and the nucleotide homology with other Hp strains on the GenBank was 94%-98%. The molecular weight of the recombinant napA-pGEX-4T-1 expressed in E.coli was 44 kDa. 3 of 29 anti-Hp mAbs were against NAP. Western blot analysis proved that the recombinant NAP was specifically recognized by the sera of Hp-infected patients.

CONCLUSION

The recombinant NAP has original immunoreaction. It is of great value to clinical sero-diagnosis and vaccine study of Hp.

摘要

目的

构建幽门螺杆菌（Hp）（中性粒细胞激活蛋白）napA基因的原核表达系统，分析核酸序列并研究其免疫性和炎症反应。

方法

采用聚合酶链反应（PCR）从Hp NCTC11639染色体DNA中扩增napA片段。将其进行T-A克隆、测序，并与GenBank上的其他Hp菌株进行比较。然后将该基因克隆到pGEX-4T-1融合表达载体中，在大肠杆菌中表达，并用谷胱甘肽-S-转移酶（GST）亲和层析法纯化。用纯化产物筛选29株小鼠抗Hp单克隆抗体（mAb），并用Hp感染患者的血清通过蛋白质印迹法分析其免疫性和炎症反应。

结果

napA片段由435个碱基对组成（GenBank编号：DQ341279），与GenBank上其他Hp菌株的核苷酸同源性为94%-98%。在大肠杆菌中表达的重组napA-pGEX-4T-1的分子量为44 kDa。29株抗Hp mAb中有3株针对NAP。蛋白质印迹分析证明重组NAP能被Hp感染患者的血清特异性识别。