幽门螺杆菌细胞毒素相关抗原A的表达纯化及其临床研究

Expression, purification and clinical research of Helicobacter pylori cytotoxin-associated gene A

目的:cagA^+Hp的感染与胃癌的相关性在亚洲有不同报道,本研究应用重组幽门螺杆菌毒素相关抗原A(CagA)建立检测血清中抗CagA抗体的方法,以探讨本地区cagA^+ Hp的感染与胃癌的相关性。 方法:构建表达幽门螺杆菌CagA抗原的表达载体,工程菌经IPTG诱导,表达的CagA包含体经Ni柱纯化,变性、复性、透析并经活性鉴定后,抗原被点在硝酸纤维素膜上或用以包被ELISA板,建立检测正常人及胃癌患者血清抗CagA抗体的DIGFA(斑点金免疫渗滤试验,简称滴金法)和ELISA法。 结果:重组克隆pRSETcagA的工程菌可表达M_r36000的目的蛋白,表达形式为包含体;CagA包含体经纯化后SDS-PAGE显示纯度达98％以上,活性经ELISA证实;正常人64例及胃癌患者血清50例中,cagA^+ Hp的感染率分别为29.7％和62％,胃癌患者cagA^+ Hp的感染率明显高于正常人(P<0.01)。 结论:我们建立的检测血清抗CagA抗体的DIGFA和ELISA均具有良好的可重复性;胃癌患者cagA^+ Hp的感染比例明显高于正常人,CagA阳性幽门螺杆菌的感染与胃癌有关,CagA可作为制备防治cagA^+ Hp感染的疫苗的后备抗原。CagA可作为幽门螺杆菌疫苗制备的候选抗原,检测患者血清幽门螺杆菌CagA抗体,有可能为胃癌的发生提供预警作用。

AIM: The relationship between cagA+ Hp infection and gastric cancer in Asia was reported inconsistently. Using recombinant cytotoxin-associated protein A (CagA) of Helicobacter pylori expressed in E. coli, we have established dot immunogold filtration assay (DIGFA) and enzyme-linked immunosorbent assay (ELISA) to detect serum anti-CagA antibody and then performed a study on the relationship between cagA+ Hp infection and gastric cancer.METHODS: After the recombinant plasmid pRSETcagA was proved, the engineered bacteria were induced to express CagA protein by IPTG, and CagA inclusions were denatured, washed through Ni-column, renatured and dialyzed. The purity and activity was revealed by Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA. Antigen was added on nitrocellulose membrane of 0.45 μm pore size or coated on ELISA plates, then anti-CagA antibodies in sera from gastric cancer patients and healthy controls were determined by DIGFA or ELISA respectively.RESULTS: The recombinant CagA antigen was about Mr36 000, revealed 98 % purity and good activity. Anti-CagA antibody was present in 19/64 (29.7 %) gastric cancer and in 31/ 50 (62 %) healthy controls. The gastric cancer subjects were more likely than the healthy controls to have a positive anti-CagA antibody assay ( P< 0.01). CONCLUSION: The newly developed anti-CagA antibody assays were highly reproducible. Expression of anti-CagA antibody was present in a significantly higher percentage of gastric cancer subjects than in healthy controls. It is suggested that cagA+ Hp infection will increase the possibility of cagA+ Hp infection subjects to develop gastric cancer. CagA can be a candidate antigen as vaccine to protect and cure cagA+ Hp infection. The detection of anti-CagA antibody in sera will provide patient early warning of developing gastric cancer in future.