抗三肽囊素单克隆抗体的制备  

Preparation of Monoclonal Antibodies against Bursin

作　　者：邓舜洲[1] 郑智刚[1] 张文波[1] 谢笑芳[1] 邹柳[1] 邬向东[1] 谌南辉[1] 何后军[1] 朱芝秀[1] 

机构地区：[1]江西农业大学动物科学技术学院,江西南昌330045

出　　处：《中国畜牧兽医》2013年第10期76-80,共5页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31160498);江西省自然科学基金(2010GZN0046);江西省教育厅项目(GJJ11402)

摘　　要：为研制抗三肽囊素(bursin,KHG-NH2)的单克隆抗体,本试验采用EDC法将三肽囊素分子赖氨酰(K)上的氨基与载体蛋白(BSA或OVA)定向偶联,分别制备三肽囊素免疫抗原(BSA-KHG-NH2)和检测抗原(OVA-KHG-NH2)。以BSAKHG-NH2免疫BALB/c小鼠,应用杂交瘤技术,筛选到18株能稳定分泌抗三肽囊素单克隆抗体的杂交瘤细胞株。这18株单克隆抗体与三肽囊素人工抗原的结合均能被三肽囊素、GKHG-NH2四肽阻断,但不能被KHGK四肽阻断,表明18株单抗与三肽囊素分子的结合具有高度特异性。以本研究制备的单抗2D2建立间接竞争ELISA方法,检测三肽囊素含量,检测下限为4ng/mL,线性范围为4～500ng/mL,加标检测回收率为85.0%～92.4%。In order to produce monoclonal antibodies（McAbs）against bursin,the artificial antigens of bursin（BSA-KHGNH2 and OVA-KHG-NH2）were prepared by EDC method,in which the amino group on-Lys of bursin molecules was conjugated to bovine serum albumin（BSA）or chicken ovalbumin（OVA）,respectively.BALB/c mice were immunized four times by BSA-KHG-NH2, and eighteen hybridoms producing anti-bursin monoclonal antibodies were obtained by limited dilution.The binding of artificial antigen OVA-KHG-NH2 to the McAbs were competitively inhibited by free bursin（KHG-NH2） and GKHG-NH2 peptide,respectively,but couldn＇t be inhibited by free KHGK peptide.These results indicated that the 18McAbs specifically bonded to bursin molecules.An IC-ELISA method to detect bursin was developed using McAb 2D2,in which the detection limits was about 4ng/mL and the linear range of the inhibition curve was about 4 to 500ng/mL,the recovery rate of bursin in the samples ranged from 85.0% to 92.4% .

关 键 词：三肽囊素 人工抗原 单克隆抗体 间接竞争ELISA 

分 类 号：S852.43[农业科学—基础兽医学]