哺乳动物促卵泡素氨基酸序列特征及其单克隆抗体的制备与鉴定

发布时间：2018-09-01 14:20

【摘要】：为制备哺乳动物促卵泡素(FSH)单克隆抗体,比对了人类、家畜、部分野生动物、鼠类等哺乳动物FSH氨基酸序列,分析其种间同源性,结果发现,多种哺乳动物FSHβ亚基氨基酸序列的同源性高于80%,且线性抗原表位高度保守。从奶牛垂体组织扩增FSHβ亚基CDS片段,构建p ET28aFSH重组质粒,诱导表达FSH重组蛋白,免疫Balb/c小鼠,取免疫小鼠的脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA法筛选阳性杂交瘤细胞孔,取效价最高孔进行克隆和亚克隆,获得1株能够分泌抗FSH的单克隆抗体细胞株,命名为B4A5。B4A5细胞株经过数次传代培养、反复冻融后生长状态良好,且能够稳定分泌抗体。采用细胞株体外培养和小鼠腹水诱生法均制备了抗FSH的单克隆抗体,其效价分别为1∶640、1∶106。分别采用Western blot和间接ELISA法鉴定单克隆抗体的结合特异性和交叉反应性,结果显示,制备的单克隆抗体与FSH反应呈阳性,与促黄体素(LH)、促甲状腺素(TSH)、促乳素(PRL)、人绒毛膜促性腺激素(h CG)均无交叉反应性。综上,获得了1株能够稳定分泌单克隆抗体的杂交瘤细胞株,其分泌的单克隆抗体可特异性结合FSH蛋白。
[Abstract]:In order to prepare mammalian follicle stimulating hormone (FSH) monoclonal antibody, the FSH amino acid sequences of human, domestic animals, some wild animals and mice were compared and their homology was analyzed. The homology of amino acid sequence of FSH 尾 subunit in many mammals was higher than that of 80, and the linear antigen epitope was highly conserved. The FSH 尾 subunit CDS fragment was amplified from the pituitary tissue of dairy cow, the recombinant plasmid of p ET28aFSH was constructed, the recombinant FSH protein was induced to express, the Balb/c mice were immunized, the spleen cells of the immunized mice were fused with SP2/0 myeloma cells, and the positive hybridoma cells were screened by indirect ELISA method. A monoclonal antibody cell line which can secrete FSH was obtained by cloning and subcloning with the highest titer. It was named as B4A5.B4A5 cell line after several passages. After repeated freezing and thawing, the cell line grew well and secreted antibody stably. Monoclonal antibodies against FSH were prepared by cell line culture in vitro and mouse ascites induction method. The titers of monoclonal antibodies against FSH were 1: 640,1: 106, respectively. Western blot and indirect ELISA methods were used to identify the binding specificity and cross-reactivity of monoclonal antibodies. The results showed that the prepared monoclonal antibodies were positive for FSH reaction. There was no cross reactivity with human chorionic gonadotropin (h CG) with luteinizing hormone (LH), thyrotropin (TSH), lactotropin (PRL),). In conclusion, a hybridoma cell strain which can secrete monoclonal antibody stably was obtained. The monoclonal antibody secreted by hybridoma could specifically bind to FSH protein.
【作者单位】：河南牧业经济学院动物医学院;
【基金】：河南省教育厅高等学校重点科研计划项目(16B230003) 河南牧业经济学院学术带头人培养计划项目(25000231)
【分类号】：S814

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