摘要
目的:构建幽门螺杆菌(Helicobacterpylori,Hp)cagA基因片段的原核表达系统,建立ELISA检测CagA及其抗体,了解表达CagA的Hp菌株(CagA+Hp)感染与所致疾病种类的关系。方法:从快速尿素酶试验阳性的156例患者胃黏膜活检标本中分离Hp。采用PCR检测109株HpcagA基因,并从临床菌株Y06中扩增2148bp的cagA基因片段(cagA1)。构建cagA1原核表达系统,用SDS-PAGE检查目的重组蛋白(rCagA1)的表达情况,用Westernblot和免疫双扩散试验鉴定rCagA1的免疫反应性和抗原性。建立ELISA,检测109株HpCagA表达和相应患者血清中CagA抗体。分析CagA+Hp感染与胃炎和消化性溃疡的关系。结果:80.8%胃黏膜活检标本中分离出Hp(126/156),97.2%的Hp菌株(106/109)cagA基因阳性。与文献报道比较,所克隆的cagA1片段核苷酸和氨基酸序列同源性分别为94.83%和93.30%。所构建的原核表达系统表达的rCagA1量约为细菌总蛋白的30.0%。rCagA1能与Hp全菌抗体发生结合反应,免疫家兔产生双扩散效价为1∶4的抗体。92.6%菌株(101/109)可表达CagA,88.1%患者(96/109)血清CagA抗体阳性。消化性溃疡标本中CagA+Hp菌株分离阳性率(97.9%)高于胃炎标本(88.5%),但无统计学差异(χ2=3.48,P>0.05)。
<Abstrcat> Objective: To construct a prokaryotic expression system of a fragment from Helicobacter pylori cagA gene and to detect the CagA positive Helicobacter pylori (CagA^+ H.pylori) and its antibody with the recombinant protein cagA 1. Methods: H.pyloriisolates were obtained from biopsy specimens of 156 patients with gastric diseases.PCR method was used to detect frequency of cagA gene in 109 H.pyloriisolates and to amplify a 2 148 bp fragment (cagA1) of cagA gene from a clinicalstrain Y06.A prokaryotic expression system of cagA1 was constructed.Expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE.Western blot andimmunodiffusion assay were applied to determine immunoreactivity and antigenicity of rCagA1.Two ELISA protocols were established to detect CagA expression in 109 H.pylori isolates and CagA antibody in serum of patients with gastric diseases.Correlations between infection of CagA^+ H.pylori and gastric diseases were analyzed. Results: H.pylori strains were isolated from 80.8% of the biopsy specimens (126/156) and 97.2% of the isolates (106/109) were cagA gene positive.In comparison with the reported data,homologies of nucleotide and putative amino acid sequences of the cloned cagA1 fragment were 94.83%and 93.30%,respectively.The output of rCagA1 was approximate 30.0% of the totalbacterial proteins.rCagA1 was able to combine with the commercial antibody against whole cell of H.pylori and to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1∶4. 92.6% of the H.pylori isolates(101/109) expressed CagA and 88.1% of serum samples (96/109) were CagA antibodypositive.The percentage of CagA^+ H.pylori strains (97.9%) of peptic ulcer trended to be higher than that of gastritis (88.5%),but there was no statisticallysignificant difference between two groups (χ~2=3.48,P>0.05). Conclusion: The recombinant rCagA1 can be used to detect CagA of H.pylori and its antibody.No association is found between CagA expression of H.pylori strains and types of gastric
出处
《浙江大学学报（医学版）》
CAS
CSCD
2005年第3期223-229,共7页
Journal of Zhejiang University(Medical Sciences)
基金
国家教育部优秀年轻教师基金.
