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

来源 :第四军医大学学报 | 被引量 : 0次 | 上传用户:fangming286
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目的  cag A+ 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 (Cag A)建立检测血清中抗 Cag A抗体的方法 ,以探讨本地区 cag A+ Hp的感染与胃癌的相关性 .方法 构建表达幽门螺杆菌 Cag A抗原的表达载体 ,工程菌经 IPTG诱导 ,表达的 Cag A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 EL ISA板 ,建立检测正常人及胃癌患者血清抗 Cag A抗体的 DIGFA(斑点金免疫渗滤试验 ,简称滴金法 )和 EL ISA法 .结果 重组克隆 p RSETcag A的工程菌可表达 Mr36 0 0 0的目的蛋白 ,表达形式为包含体 ;Cag A包含体经纯化后 SDS- PAGE显示纯度达 98%以上 ,活性经 EL ISA证实 ;正常人 6 4例及胃癌患者血清 5 0例中 ,cag A+ Hp的感染率分别为 2 9.7%和6 2 .0 % ,胃癌患者 cag A+ Hp的感染率明显高于正常人 (P<0 .0 1) .结论 我们建立的检测血清抗 Cag A抗体的 DIGFA和EL ISA均具有良好的可重复性 ;胃癌患者 cag A+ Hp的感染比例明显高于正常人 ,Cag A阳性幽门螺杆菌的感染与胃癌有关 ,Cag A可作为制备防治 cag A+ Hp感染的疫苗的后备抗原 . Cag A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 Cag A抗体 ,有可能为胃癌的发 Objective cag A + Hp infection and gastric cancer have been reported in Asia. In this study, recombinant adenovirus-associated antigen A (Cag A) was used to detect anti-Cag A antibody in serum to explore the relationship between cag A + Hp .Methods The expression vector of Cag A antigen of Helicobacter pylori was constructed and induced by IPTG. The expressed Cag A inclusion body was purified, denatured, refolded and dialyzed by Ni column, The antigen was spotted on nitrocellulose membrane or used to coat the EL ISA plate to establish a DIGFA (gold-immunodialysis assay) method for detecting anti-Cag A antibodies in normal and gastric cancer patients and the EL ISA method. Results The recombinant plasmid pRSETcag A could express the target protein of Mr3600 in the form of inclusion body. The purity of Cag A inclusion body was over 98% by SDS-PAGE and the activity was confirmed by ELISA. The normal human The prevalences of cag A + Hp in 26 cases of gastric cancer and 50 cases of gastric cancer were 2. 9.7% and 6.2%, respectively. The infection rates of cag A + Hp in gastric cancer were significantly higher than those in normal people (P <0.01) Conclusion We set up The detection of serum anti-Cag A antibody DIGFA and EL ISA have good repeatability; gastric cancer patients cag A + Hp infection was significantly higher than normal, Cag A positive Helicobacter pylori infection and gastric cancer, Cag A can be used as preparation Cag A can be used as a candidate antigen in the preparation of H. pylori vaccine for the detection of H. pylori Cag A antibody in patients with gastric cancer
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