斑马鱼cdk7和cyclinH基因的原核表达及多克隆抗体的制备与鉴定  

Prokaryotic Expreesion and Polyclonal Antibodies Preparation of cdk7 and cyclin H in Zebrafish

作　　者：储琳[1] 刘清云[1] 钱旻[2] 严缘昌[1] 李逸平[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031 [2]华东师范大学生命科学学院,上海200062

出　　处：《细胞生物学杂志》2006年第6期849-854,共6页Chinese Journal of Cell Biology

基　　金：上海教育委员会E-研究院建设计划项目资助(No.E03003)~~

摘　　要：构建了两个表达斑马鱼cdk7(基因片段)和cyclinH(基因全长)的重组质粒,分别转化大肠杆菌BL21进行原核表达,所得的CDK7和cyclinH两个融合蛋白均以包涵体形式存在。经SDS-PAGE分离,切下含有目的蛋白条带的凝胶冻成干粉,分别免疫新西兰大耳兔,制备并纯化了分别抗CDK7和抗cyclinH的两个多克隆抗体。经酶联免疫吸附测定、蛋白质印迹分析检测,确定获得了两种具有较高效价的特异性多克隆抗体。免疫荧光组织化学结果显示,CDK7和cyclinH这两个蛋白质均普遍存在于斑马鱼胚胎动物极的各个细胞中。Expression vectors of CDK7 and cyclin H gene in zebrafish were constructed and transformed into E. coli BL21 separately for IPTG induced expression. The results showed that both fusion protein were expressed in the form of inclusion body. Fusion proteins were isolated and then lyophilized to raise the polyclonal antisera against CDK7 and cyclin H respectively by immunizing New Zealand rabbits. The antisera were purified and characterized by ELISA, Western blotting and immunofluorescence-staining. The results showed that the antibodies had high titer, affinity and specificity.

关 键 词：斑马鱼 CDK7 CYCLIN H 原核表达 抗血清 

分 类 号：Q78[生物学—分子生物学]