幽门螺杆菌UreB单克隆抗体的制备及尿素酶活性抑制能力的研究  

Preparation of anti-Helicobacter pylori UreB monoclonal antibody and the detection of the ability of inhibition the urease activity

作　　者：吴亚男[1] 邹全明[1] 罗萍[1] 郭刚[1] 王晓勤[1] 

机构地区：[1]第三军医大学临床微生物学及免疫学教研室重庆市生物制药工程技术研究中心,重庆400038

出　　处：《免疫学杂志》2005年第5期393-396,共4页Immunological Journal

基　　金：国家"九五"重点科技攻关项目(96-901-01-54);全军"九五"医药卫生科研基金项目(98D044)资助

摘　　要：目的制备抗幽门螺杆菌尿素酶B亚单位(UreB)单克隆抗体,并检测其对尿素酶活性的抑制能力。方法以重组UreB蛋白为免疫原,通过杂交瘤技术制备抗UreB单克隆抗体,用ELISA检测mAb的效价、亲和常数;用Westernblot检测mAb的特异性。将尿素酶与单克隆抗体预孵育后加入含有尿素和酚红的缓冲液,于550nm测定单克隆抗体对尿素酶活性的抑制能力。结果得到5株稳定分泌抗UreB的单克隆抗体的杂交瘤细胞1D5、9E1、3A2、1D6、6E6。测定抗体亚型1D5,9E1,3A2为IgG1,1D6、6E6为IgG2a,亲和常数分别为4×108、4.6×108、2.8×108、2×109、3×109L/mol。Westernblot鉴定5种单克隆抗体均能和重组UreB及全菌蛋白中的UreB抗原发生特异性结合,且均不与肠道常见菌发生交叉反应。5株单克隆抗体中只有6E6具有对尿素酶活性的抑制作用,且抑制率与单克隆抗体剂量相关。结论制备了5株稳定分泌抗UreB抗体的杂交瘤细胞株,产生的单克隆抗体效价高且特异性好,其中6E6能抑制尿素酶活性。本研究为探讨尿素酶的作用机制、UreB的纯化及Hp的临床诊断和治疗奠定了基础。Objective To prepare the anti-UreB monoclonal antibody （mAb）, and detect the inhibition ability of the anti-UreB mAb on urease activity. Methods mAb was prepared by hybridoma technique using recombined UreB （rUreB） as i mmunogen. The titer and affinity constant of the mAb were determined by ELISA and the specificity of the mAbs was analyzed by Western blotting. The mAb was preincubated with the urease fraction, and then the buffer containing urea and phenol red was added into the culture solution. The inhibition ability of mAb on urease activity was detected at 550 nm. Results Five hybridoma cell lines of 1D5, 9E1, 3A2, 1D6, 6E6 were screened, which could secrete the anti-UreB mAb stably. The 5 mAbs have isotypes of IgG1/IgG1/IgG1/IgG2a/IgG2a, respectively, and affinity constant of 4×10^8, 4.6×10^8, 2.8×10^8, 2×10^9, 3×10^9L/mol, respectively. Each of the 5 antibodies could bind with rUreB or the UreB antigen specifically, and could not cross-react with bacteria protein in intestinal tract. Among 5 mAbs, only 6E6 could inhibit the activity of urease in a dose-dependent manner. Conclusion Five hybridoma cell strains are prepared, which can secrete high-titer and high-specific monoclonal antibodies against UreB stably, and one mAb （6E6） of them can inhibit the urease activity. The result provides a basis for the mechanism research of urease and affords a valuable tool for UreB purification as well as Hp diagnose and clinical treatment.

关 键 词：尿素酶B亚单位 单克隆抗体 尿素酶活性 

分 类 号：R392[医药卫生—免疫学]