Preparation,identification and application of monoclonal antibody against urease subuint B of Helicobacter pylori

目的制备抗幽门螺杆菌(HP)的单克隆抗体(MAb),并对该HP MAb进行分析鉴定,建立一种检测患者由于HP感染而在血清中产生HP抗体的竞争ElISA检测方法。方法用灭活的HP免疫bAlb/C小鼠,通过杂交瘤技术制备HP MAb。我们采用HP混合蛋白包括毒素相关蛋白A(CAgA)、空泡毒素A(VACA)和尿素酶以及灭活的HP菌体筛选阳性杂交瘤细胞株,用ElISA和WESTErn blOT等技术对所获得的HP MAb进行鉴定。利用辣根过氧化物酶(HrP)标记所筛选的HP MAb来建立一个可检测患者血清中HP抗体的竞争ElISA。结果通过大规模的杂交瘤筛选,我们选择了1株将其命名为C3 HP MAb,其抗体亚型为Igg2A,腹水效价可达1x107。WESTErn blOT法、ElISA和质谱检测结果显示,该C3 HP MAb能特异地识别HP的尿素酶b亚单位。用这个C3 HP MAb,我们建立了一种可检测患者血清中HP抗体的竞争ElISA。结论成功获得一种可以特异识别HP尿素酶b亚单位的MAb,建立了一种可检测患者血清中HP抗体的竞争ElISA。Objective To prepare and characterize the monoclonal antibody(mAb) against Helicobacter pylori(Hp)and establish a competitive ELISA used for detection of Hp antibodies in the sera of Hp-infected patients.Methods BALB /c mice were immunized with inactivated Hp to generate Hp mAb using the hybridoma technology.Hp mixed proteins including cytotoxin-associated gene A(CagA),vacuolating cytotoxin A(VacA) and urease,as well as inactivated Hp were applied to screen positive hybridoma.Selected Hp mAb was analyzed and characterized with ELISA and Western blotting,and then labeled with horseradish peroxidase( HRP) for establishing a competitive ELISA to detect Hp antibodies in the sera of Hp-infected patients.Results One Hp mAb named as C3 was selected after screening large amount of hybridoma,and the C3 Hp mAb was special for IgG2 a subtype with the affinity titer of 1 × 107.Western blotting,ELISA and mass spectrum analysis indicated that the C3 Hp mAb could recognize Hp urease subunit B specifically.Using the C3 Hp mAb,we developed a competitive ELISA which could be used to detect Hp antibodies in the sera of Hp-infected patients.Conclusion We successfully obtained one mAb that could specifically recognize Hp urease subunit B and developed a competitive ELISA using the Hp mAb.福建省科技重点项目(2011Y0050); 厦门市科技计划项目(3502Z20123009); 国家基础科学人才培养基金(J1310027

卫矛科沟瓣属与卫矛属的数量分类学研究

卫矛属(Euonymus L.)和沟瓣属(Glyptopetalum Thw.)的系统关系长期以来备受争议,为理清它们之间的关系,该研究选择沟瓣属12种、卫矛属17种和假卫矛属(Microtropis Wall. ex Meisn.) 2种,共计31个分类单元(OTUs),测量并统计了56个形态性状,同时利用聚类分析(UPGMA)和主坐标分析(PCoA)的方法进行了数量分类学研究。结果表明:沟瓣属与卫矛属具有较高的表征相似性,二者之间存在的过渡类群有缙云卫矛(Euonymus chloranthoides Yang)和淡绿叶卫矛(E. pallidifolium Hayata)以及海南沟瓣[Glyptopetalum fengii (Chun&F. C. How) Ding Hou]、白树沟瓣[G. geloniifolium (Chun&F. C. How)C. Y. Cheng]。该研究认为将沟瓣属作为卫矛属的一个组,即沟瓣组(Sect. Glyptopetalum),放在冬青卫矛组(Sect. Ilicifolia)附近更为合理。对性状的主坐标排序分析显示,叶片长度、叶片宽度、叶侧脉数目、花瓣数目、心室数目、每室胚珠数、花序分枝数、是否具有假种皮、蒴果是否开裂、花期与果序梗长度在卫矛属分类中具有重要的价值