大熊猫LIF基因的原核表达及其单克隆抗体的制备与鉴定  

Prokaryotic expression of LIF gene of giant panda and preparation and characterization of monoclonal antibodies

作　　者：刘项宇 张梦诗 李非平 王神飞 胡贤彪 刘玉良 侯蓉 蔡开来 LIU Xiangyu;ZHANG Mengshi;LI Feiping;WANG Shenfei;HU Xianbiao;LIU Yuliang;HOU Rong;CAI Kailai(Sichuan Provincial Key Laboratory of Endangered Wildlife Conservation Biology,Chengdu Research Base of Giant Panda Breeding,Chengdu 610081,China)

机构地区：[1]成都大熊猫繁育研究基地四川省濒危野生动物保护生物学重点实验室,四川成都610081

出　　处：《中国兽医科学》2024年第5期624-631,共8页Chinese Veterinary Science

基　　金：四川省自然科学基金重点项目(2022NSFSC0010);成都大熊猫繁育研究基地自立课题(2022CPB-B10);成都大熊猫繁育研究基地自立课题(2024CPB-P02)。

摘　　要：为获得大熊猫LIF单克隆抗体,将大熊猫LIF基因编码区23~203基因片段添加酶切位点合成后连接到pET30a原核表达载体上,转化BL21感受态细胞,经IPTG诱导表达、纯化蛋白后作为免疫原,对BALB/c小鼠进行免疫,并按照常规方法制备杂交瘤细胞,应用间接ELISA方法筛选鉴定阳性杂交瘤细胞株。结果显示,成功构建了LIF(23-202)-pET30a重组质粒,获得了高纯度的LIF重组蛋白免疫原,得到1株能分泌大熊猫LIF单克隆抗体的杂交瘤细胞株,其分泌的抗体亚型为IgG2b。Western-blot结果显示,LIF-2单克隆抗体可特异性识别大熊猫子宫内膜间质细胞、睾丸支持细胞、皮肤细胞、脐带间充质干细胞及骨髓间充质干细胞中的LIF蛋白。上述结果为阐明LIF的定位和组织表达信息,探讨LIF在大熊猫胚胎着床中的作用奠定了重要基础。In order to obtain the giant panda LIF monoclonal antibody,the coding region 23—203 gene fragment of giant panda LIF gene was synthesized by adding enzyme cutting sites and ligated into pET30a prokaryotic expression vector,the recombinant plasmid was transformed into BL21 competent cells,the protein was induced by IPTG and purified as an immunogen to immunize BALB/c mice.The hybridoma cells were prepared by conventional method,and the positive hybridoma cell lines were screened and identified by indirect ELISA.The results showed that the LIF(23-202)-pET30a recombinant plasmid was successfully constructed,recombinant LIF protein immunogen with high purity was obtained.A hybridoma cell line secreting giant panda LIF monoclonal antibody was obtained,and the antibody subtype was IgG2b.The results of Western-blot showed that LIF-2 monoclonal antibody could specifically detect LIF protein in endometrial stromal cells,testicular Sertoli cells,skin cells,umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells of giant panda.These results laid an important foundation for elucidating the localization and tissue expression information of LIF and exploring the role of LIF in the implantation of giant panda embryos.

关 键 词：大熊猫 LIF 单克隆抗体 杂交瘤细胞 免疫印迹 

分 类 号：S857.2[农业科学—临床兽医学]