幽门螺杆菌cagAM1′基因表达抗原的纯化及应用  

Purification and application of antigens expressed by the cagAM1'gene of Helicobacter pylori

作　　者：张静[1] 陈月秀[2] 佘菲菲[2] 

机构地区：[1]福建医科大学附属协和医院老年医学研究所,福州350001 [2]福建医科大学,福州350004

出　　处：《中国人兽共患病杂志》2005年第6期495-497,共3页Chinese Journal of Zoonoses

基　　金：福建省科技项目基金(99-Z-159)

摘　　要：目的克隆cagAM1′基因,以其表达的蛋白为抗原,通过血清学方法检测幽门螺杆菌(Hp)cagA阳性菌株的感染。方法以既往克隆的cagAM1基因为模板设计引物,PCR扩增681bpcagAM1′基因,将其定向连接入载体pET-42a(+),转化宿主菌BL21,用IPTG诱导融合蛋白表达。可溶部分的表达产物用GST·Tag方法纯化。以该纯化抗原经WesternBlot方法,检测39份Hp培养阳性(其中28份cagA基因阳性,11份阴性)及17份无Hp感染患者的血清,以鉴定抗原检测的敏感性和特异性。同时用进口试剂盒进行对照实验。结果获得62kD的CagAM1′与GST融合蛋白,以此为抗原,28株cagA阳性患者的血清26份检出抗cagA抗体,表明cagAM1′蛋白具有抗原性,且该抗原检测相应抗体的敏感性为92.9%,高于进口试剂盒(71.4%),17份无Hp感染患者的血清14份抗cagA抗体阴性,表明该抗原检测相应抗体的特异性为82.4%,高于进口试剂盒(52.9%)。结论cagAM1′蛋白有望进一步开发用于临床Hp高毒株感染诊断。The cloning of the Helicobacter pylori(Hp) cagAM1'gene and its application of the expressed antigen for the detection of the cagA positive strain of Hp were underwent in this study. The primers used were designed according to the cloned cagAM1'gene and used as templates, meanwhile the cagAM1'gene was amplified by PCR , inserted into the expression vector pET-42a(+) and translocated to the host strain BL21 cells after induction with IPTG to express the fusion protein. The soluble fraction was purified by GST tag method. To appraise the sensitivity and specificity in Western blot assay,39 culture-positive sera, in which 28 sera were cagA gene positive and 11 sera were negative, and 17 culture negative sera were collected and used for testing with the purified antigen.The imports commercial kit was also unparallel testing for comparison. A fusion protein containing the GST domain with a molecular mass of 62 kDa was obtained in the present study ,and it was used as antigen for testing. The anti-CagA antibody was detected from 26 sera of 28 cagA positive patients, indicating the antigenicity of the cagAM1'protein.The sensitivity of this protein to detect the corresponding antibody was 92.9%,higher than that of the commercial kit(71.4%). In 14 sera of the 17 sera of patients without Hp infection, the anti-cagA antibody was absent. The specificity of this testing was 82.4%,which was higher than that of the commercial kit (52.9%). It is concluded that cagAM1'protein can be used as an antigen for the detection of specific antibody in patients with high virulent Helicobacter pylori infection.

关 键 词：幽门螺杆菌 CAGA 血清学诊断 

分 类 号：R378.2[医药卫生—病原生物学]