幽门螺杆菌caga蛋白的纯化与鉴定.docVIP

幽门螺杆菌CagA蛋白的纯化与鉴定【 摘要 】　目的　纯化幽门螺杆菌（Hp）相对分子质量（Mr）为130×103(CagA)蛋白，为单抗及疫苗的研制打下一定基础。方法　 用盐酸胍粗提、分子筛层析及凝胶洗脱法纯化Hp97002株Mr为130×103（CagA）蛋白并鉴定。用Western blot方法分析68例患者血清中Mr为130×103（CagA）蛋白的阳性率。结果　纯化蛋白Mr为130×103，电泳纯，并保持良好的抗原性。有43例患者血清能与纯化的Mr为130×103（CagA）蛋白反应，其阳性率随着浅表性胃炎、萎缩性胃炎、消化性溃疡和胃癌的排列次序呈逐渐增高趋势。结论　Mr为130×103（CagA）蛋白与胃部疾病的严重性相关。 Purification and identification of CagA protein of Helicobacter pylori YE Shaojing, FANG Pingchu, LI Chao 　　(long. College of Medicine, Zhejiang University, Hangzhou 310031, P.R.China) 　　【 Abstract 】　Objective　To purify and identify the CagA (cytotoxin-associated gene A) protein of clinical isolate Hp97002 and to investigate the relationships between the purified CagA (molecular weight of 130000) protein and gastric diseases.Methods　Using 6M guanidine extract, size exclusion chromatography and elusion from gel was the purified protein (CagA). Sera of 68 patients with gastric diseases (44 with chronic gastritis, 15 with atrophic gastritis, 7 with peptic ulcer disease, 2 with gastric cancer) were obtained, and the serological response to CagA was studied by Western blot using the purified protein.Results　The purified protein was molecular weight of 130000 with good antigenicity and revealed basic isoelectric point about 8.1. Among 68 sera, 43 sera could recognize the purified proteins from the patients of chronic gastritis 47.7% (21/44), atrophic gastritis 86.7% (13/15), peptic ulcer disease 100% (7/7), gastric cancer 100% (2/2), the difference was significant (χ2=13.327, P=0.004).Conclusion　CagA (molecular weight of 130000) protein was associated with severe gastric diseases. 　　【 Subject words 】　Helicobacter pylori; CagA; Elution from gel; Western blot 　　幽门螺杆菌（Helicobacter pylori, Hp）感染已被公认为人类慢性胃炎、胃溃疡、十二指肠溃疡的重要病因，并且与胃癌和胃粘膜相关淋巴组织淋巴瘤的发生密切相关。全球范围内大约有50%～80%的成人有Hp感染〔1〕。但Hp感染人群大多无症状，只有少数感染者可出现胃溃疡、十二指肠溃疡，极少数感染者最终发展为胃癌。出现上述不同感染结果的主要原因是Hp菌株的多样性〔2，3〕。国外目前的研究表明，Hp菌株cagA (cytotoxin-associated gene A)及其表达产物CagA蛋白与胃部疾病的严重性相关〔4-6〕，但CagA蛋白的功能迄今尚不清楚。本实验采用膜蛋白的分离纯化原理和技