南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (04): 425-427.

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抗幽门螺杆菌单克隆抗体的制备与鉴定

李妍； 宁云山； 洪燕华； 刘宜楚； 罗军； 龙敏； 董文其； 李明；   

  1. 南方医科大学生物技术学院； 南方医科大学公共卫生与热带病学院微生物系； 南方医科大学生物技术学院 广东 广州 510515； 广东 广州 510515；
  • 出版日期:2006-04-20 发布日期:2006-04-20
  • 基金资助:
    国家重大攻关项目(2001CB510208)~~

Preparation and identification of monoclonal antibodies against Helicbacter pylori

LI Yan, NING Yun-shan, HONG Yan-hua, LIU Yi-chu, LUO Jun, LONG Min, DONG Wen-qi, LI Ming School of Biotechnology, Department of Microbiology, School of Public Health and Tropical Disease, Southern Medical University, Guangzhou 510515, China   

  1. 南方医科大学生物技术学院； 南方医科大学公共卫生与热带病学院微生物系； 南方医科大学生物技术学院 广东 广州 510515； 广东 广州 510515；
  • Online:2006-04-20 Published:2006-04-20

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摘要： 目的制备抗幽门螺杆菌(Helicobacter pylori,Hp)全菌的单克隆抗体并对其特异性进行初步鉴定。方法用培养的幽门螺杆菌超声破菌后的上清和沉淀为抗原分别免疫BALB／c小鼠,通过杂交瘤技术制备单克隆抗体并测定抗体免疫球蛋白亚类、效价及亲和力,用重组表达的Hp Lpp20、HspA、urease A、CagA、urease B、catalase蛋白鉴定抗体。结果共获得34株抗Hp的单克隆抗体,其中抗上清31株和抗沉淀3株,34株抗体中针对Lpp20、HspA、urease A、CagA、urease B、catalase蛋白的抗体分别为3、2、4、1、5、2株,目前已鉴定的部分单克隆抗体的亚类均为IgGl,细胞培养上清的效价为1:16-1:32,腹水效价为1:32000～1:64000,抗体亲和常数介于1×10-10mol／L-5．2×10-12mol／L。结论获得特异性针对幽门螺杆菌的单克隆抗体,为幽门螺杆菌的诊断和疫苗研究奠定基础。 

Abstract: Objective To prepare and identify monoclonal antibodies (mAbs) against Helicobacter pylori (Hp). Methods BALB/c mice were immunized with the supernatant and precipitation of cultured Hp after ultrasonication and mAbs were obtained by means of hybridoma technique. The resultant mAbs was evaluated for subtype, titer, affinity, and further identified with Lpp20, HspA, urease A, CagA, urease B, and catalase prepared by recombinant expression. Results Totally 34 hybridoma cell lines were established which secreted specific mAbs, including 31 against the supernatant and 3 against the precipitation of Hp, and the prepared mAbs showed specific reaction against Lpp20 (3 strains), HspA (2 strains), urease A (4 strains), CagA (1 strain), urease B (5 strains), and catalase (2 strains) antigens, respectively. The mAbs was all identified as immunoglobulin G1 (IgG1) and theirs titer in the culture supernatant and ascites was 1:16 to 1:32 and 1: 32000 to 1:64000 respectively with affinity constants (Kaff) ranging from 1×10-10 to 5.2×10-12 mol/L. Conclusion The mAbs specially against Hp have been obtained, which may facilitate further study of detection and vaccine development of Hp. 

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