草鱼促性腺激素基因的原核表达及多克隆抗体的制备-生命科学研究.PDFVIP

17 卷 4 期 生命科学研究 灾燥造援员7 晕燥援4 圆园13 年8 月 蕴蚤枣藻杂糟蚤藻灶糟藻 砸藻泽藻葬则糟澡 Aug. 圆园13 草鱼促性腺激素基因的原核表达 及多克隆抗体的制备 王艳杰, 刘 琼, 陈文娟, 周 毅, 刘少军, 李建中* (湖南师范大学生命科学学院, 中国湖南 长沙4 10081) 摘 要: 选用原核表达载体pGEX鄄4T鄄1, 分别插入草鱼GTH 和FSH茁 的cDNA 序列, 构建成N 端含有GST 融 合蛋白标签的表达质粒, 分别转化大肠杆菌BL21 (DE3), 在IPTG 诱导下表达出2 个融合蛋白, SDS鄄聚丙烯酰 胺凝胶电泳显示重组融合蛋白GST鄄GTH 和GST鄄FSH茁 的相对分子质量大约为35 38 kD. 用抗GST 标签的单 克隆抗体分别对2 个表达蛋白进行Western blot 鉴定, 结果显示重组蛋白表达正确. 利用制备型SDS鄄PAGE 纯 化回收的蛋白并免疫新西兰大白兔, 分别制备了抗GTH 和抗FSH茁 的具有较高效价的多克隆抗体. 该结果为 纯化天然GTH 蛋白提供了有效的检测手段, 也为进一步制备GTH 单克隆抗体奠定了基础. 关键词: 草鱼; 促性腺激素; 基因克隆; 原核表达; 多克隆抗体 中图分类号: Q789 文献标识码: A 文章编号: 1007鄄7847(20 13)04鄄0321鄄04 Prokaryotic Expression and Preparation of Polyclonal Antibody of Gonadotropins in Ctenop hary ngodon idella WANG Yan鄄jie, LIU Qiong, CHEN Wen鄄juan, ZHOU Yi, LIU Shao鄄jun, LI Jian鄄zhong* (College of L ife Sciences, H unan Normal Unive rs ity, Changsha 4 10081, H unan, China) Abstract ：Two type of cDNA clones of GTHs , GTH and FSH subunits were inserted into PGEX鄄4T鄄1 vec鄄 tors. The recombinant plasmids of GTHs were constructed as N鄄terminal GST鄄tagged fusion proteins and then transformed into E. coli BL21. The results of SDS鄄PAGE showed that the two recombinant fusion pro鄄 teins with relative molecular mass of 35 kD and 38 kD were efficiently expressed by IPTG induction. The results of western鄄blotting suggested that the recombinant proteins were expressed correctly, just as expected. The polyclonal antibodies were prepared by immunizing New Zealand rabbits with purified fusion proteins and p