阪崎肠杆菌多克隆抗体的制备与鉴定

doi:10.11924/j.issn.1000-6850.casb17090123

中国农学通报 ›› 2018, Vol. 34 ›› Issue (30): 123-128.doi: 10.11924/j.issn.1000-6850.casb17090123

所属专题：生物技术

阪崎肠杆菌多克隆抗体的制备与鉴定

焦秋伟,孙欢欢,孟利

黑龙江大学生命科学学院分子生物学重点实验室,黑龙江大学生命科学学院分子生物学重点实验室,黑龙江大学生命科学学院分子生物学重点实验室

收稿日期:2017-09-24 修回日期:2018-09-30 接受日期:2017-11-24 出版日期:2018-10-31 发布日期:2018-10-31
通讯作者: 孟利
基金资助:
黑龙江省普通高等学校分子生物学重点实验室（黑龙江大学）开放课题“婴儿奶粉生产线阪崎阪肠杆菌污染溯源分析及控制”(1201)；哈尔滨市科技创新人才研究专项资金项目“西藏灵菇调节2 型糖尿病小鼠菌群关键菌株研究”(2015RQQXJ042)。

Preparation and Identification of Cronobacter sakazakii Polyclonal Antibody

Received:2017-09-24 Revised:2018-09-30 Accepted:2017-11-24 Online:2018-10-31 Published:2018-10-31

摘要/Abstract

摘要： 为制备高效价的阪崎肠杆菌单克隆抗体,分别以福尔马林灭活的阪崎肠杆菌(Cronobacter sakazakii)菌体和菌体破碎细胞为免疫原免疫雌性清洁级大白兔,制备抗C.sakazakii多克隆抗体,获得的抗体效价分别为256000和64000,选取菌体免疫原制备抗体。采用辛酸-硫酸铵法纯化抗体,抗体纯度用变性聚丙烯酰胺凝胶电泳鉴定。斑点杂交法检测抗体特异性,所获抗体与S.aureus, L.monocytogenes, S.typhimurium, S.flexneri, E.coli无交叉反应,与沙门氏菌有轻微交叉反应,采用杂菌抗原反向吸附法去除了该交叉反应。选取菌体免疫原免疫动物,杂菌抗原反向吸附法纯化制备获得了对C.sakazakii特异性高的多克隆抗体,为阪崎肠杆菌免疫学检测奠定了基础。

关键词: 甘薯, 甘薯, 品种, 高淀粉, 育种

Abstract: To obtain high titerCronobacter sakazakii antibody,C. sakazakii polyclonal antibody was prepared in rabbits using formalin- inactivated C. sakazakii and C. sakazakii cell debris as antigen. The titers of antiserum were 256000 and 64000, respectively, and formalin-inactivatedC.sakazakii antigen was chosen to prepare the antibody. Antibody was purified by octanoic acid-ammonium sulfate precipitation method, the purity of the pAb was detected by SDS- PAGE. The antibody had no cross reaction with S.aureus , L. monocytogenes ,S.typhimurium ,S.flexneri andE.coli by dot blot hybridization method butsalmonella . To get rid of the disturb ofsalmonella , the purified pAb was dealt withsalmonella bacterial antigens with reverse adsorption method to eliminate the cross reaction withsalmonella . The high-titer pAb special toC.sakazakii could be obtained by formalin-inactivated antigen and purified by reverse adsorption method and it was a good foundation for immune detection method development.

中图分类号:

TS207.4

焦秋伟,孙欢欢,孟利. 阪崎肠杆菌多克隆抗体的制备与鉴定[J]. 中国农学通报, 2018, 34(30): 123-128.

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阪崎肠杆菌多克隆抗体的制备与鉴定

Preparation and Identification of Cronobacter sakazakii Polyclonal Antibody

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