草鱼促性腺激素基因的原核表达及多克隆抗体的制备

Prokaryotic Expression and Preparation of Polyclonal Antibody of Gonadotropins in Ctenopharyngodon idella

选用原核表达载体pGEX-4T-1,分别插入草鱼GTH和FSHβ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-聚丙烯酰胺凝胶电泳显示重组融合蛋白GST-GTHα和GST-FSHβ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-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FSHβ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.

Two type of cDNA clones of GTHs, GTH and FSH subunits were inserted into PGEX-4T-1 vectors. The recombinant plasmids of GTHs were constructed as N-terminal GST-tagged fusion proteins and then transformed into E. coli BL21. The results of SDS-PAGE showed that the two recombinant fusion proteins with relative molecular mass of 35 kD and 38 kD were efficiently expressed by IPTG induction. The results of western-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 proved to be specific against GTH and FSH protein respectively. These results laid a foundation for the detection and purification of natural GTH as well as their monoclonal antibody preparation.