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。 Objective: The relationship between cagA ~ + Hp infection and gastric cancer has been reported differently in Asia. In this study, we established a method for the detection of anti-CagA antibody in serum using recombinant CagA to detect the cagA ~ Hp infection and gastric cancer. Methods: The expression vector expressing CagA antigen of Helicobacter pylori was constructed. The engineered bacteria were induced by IPTG. The expressed CagA inclusion bodies were purified by Ni-column, denatured, refolded and dialyzed. After the activity was identified, the antigen was spotted on the nitrocellulose membrane Or used to coat the ELISA plate to establish a DIGFA (gold-plated immunodialysis assay) for the detection of serum anti-CagA antibodies in normal and gastric cancer patients and an ELISA method. Results: The recombinant protein of pRSETcagA could express the target protein of M_r36000 in the form of inclusion body. The purity of CagA inclusion body was over 98% by SDS-PAGE after purification. The activity was confirmed by ELISA. The normal and the gastric cancer patients In 50 cases, the infection rates of cagA ~ + Hp were 29.7% and 62%, respectively. The infection rate of cagA ~ + Hp in gastric cancer patients was significantly higher than that in normal people (P <0.01). CONCLUSION: We established the DIGFA and ELISA for the detection of serum anti-CagA antibodies with good repeatability. The infection rate of cagA ~ + Hp in gastric cancer patients was significantly higher than that of normal people. The infection of CagA-positive H. pylori was related to gastric cancer, As preparation of vaccine against cagA ~ + Hp challenge antigen. CagA can be used as a candidate antigen for the preparation of Helicobacter pylori vaccine, detection of serum Helicobacter pylori CagA antibody, may provide an early warning of the occurrence of gastric cancer.