摘要
目的：构建含人幽门螺杆菌（Hpylori，Hp）过氧化氢酶（catalase，KatA）编码基因的重组质粒，测定、分析其核酸序列，并在E.coli中表达，研究其抗原性。方法：应用PCR技术从HpDNA染色体中扩增KatA编码基因片段，将其T-A克隆和测序，并与GenBank公布的其他Hp菌株的基因序列比较，再将目的基因插入至融合表达载体pGEX-4T-1中进行表达，用GST亲和层析对其进行纯化。纯化产物用于对29株小鼠抗Hp-全菌单克隆抗体（mAb）的鉴定及与Hp啊感染患者血清进行Western blot。结果：KatA基因全长为1 515 bp，并在GenBank上登录（No．DQ333889），与GenBank公布的其他Hp菌株的核酸的同源性为96％～97％，表达的KatA融合蛋白的相对分子质量（Mr）为85 000，29株小鼠抗Hp全菌mAb中有4株mAb是针对KatA的，表达产物可被Hp感染患者的血清特异性识别。结论：重组KatA具有较好的抗原性，为坳检测试剂和疫苗的研究奠定了基础。
AIM： To construct the recombinant plasmid containing catalase（KatA） of Helicobacter pylori（ Hp）, analyze its nucleic acid sequence, express it in E. coil and study its antigenicity. METHODS： KatA fragments were amplified from Hp chromosomal DNA by PCR. Its T-A was cloned, sequenced and compared with other HP strains on the GenBank. Then the gene cloned into pGEX-4T-1 fusion expression vector was expressed in E. coil and purified by GST-affinity chromatography. The purified product was used to identify 29 stains of mouse anti Hp monoclonal antibodies and analyze antigenicity with serum of Hp-infected patients by Western blot. RESULTS： KatA fragments were composed of 1 515 bp （GenBank No. DQ333889） and the nucleotide homology with other Hp strains on the GenBank was 96% -97% . 85 kDa of the recombinant KatA-pGEX-4T-1 was expressed in E. coil 4 of 29 anti-Hp mouse monoclonal antibodies were against KatA. Western blot analysis proved that KatA was specifically recognized in the serum of Hp-infected patients. CONCLUSION： The recombinant KatA has original antigenicity. It is of great value to clinical sero-diagnosis and vaccine study of Hp.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第4期440-442,446,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重大攻关项目资助(2001CB510208)
