斑马鱼ANF基因的克隆及多克隆抗体的制备  

Cloning and Polyclonal Antibody Preparation of ANF Gene in Zebrafish

作　　者：李竞超[1] 吴坚[1] 梁艳[1] 莫小阳[1] 吴秀山[1] 

机构地区：[1]湖南师范大学蛋白质化学及鱼类发育生物学教育部重点实验室心脏发育研究中心,湖南长沙410081

出　　处：《激光生物学报》2011年第5期645-649,共5页Acta Laser Biology Sinica

基　　金：国家自然科学基金项目(No.31071999;30771 170;30971 105;30971663;30871340;30671053;30970425;30900851);湖南省自然科学基金项目(10JJ1006)

摘　　要：心房钠尿肽ANF是钠尿肽家族中的一员,是心肌细胞分泌的一种循环激素,主要在心房组织分泌。生物信息学分析表明,斑马鱼ANF基因开放阅读框全长321bp,编码106个氨基酸,PCR扩增获得斑马鱼ANF基因全长。将所得的PCR片段插入原核表达载体pGEX-4T-1中,并将重组质粒(pGEX-4T-1-ANF)转化大肠杆菌BL21。通过IPTG诱导表达GST-ANF融合蛋白,经GST亲和层析法纯化后,免疫新西兰大白兔制备了多克隆抗体,Western blotting分析表明,制备的抗体具有良好的高效价性和特异性。Atrial natriuretic peptide ANF is one member of natriuretic peptide family, which is a circulation excitatory autacoid secreted by myocardial cell, mainly secrete in cardiac atrium. Zebrafish ANF gene open reading frame has 321 bp, encodes 106 amino acids. The full-length of ANF gene in zebrafish was cloned by bioinformatics combined with PCR technique. We inserted the PCR fragment into prokaryotie expression vector pGEX-4T-1, and then transformed the recombinant plasmid （pGEX-4T-1-ANF） into BL21, The GST-ANF fusion protein is induced by IPTG. The purified GST- ANF fusion protein is used to immune the New Zealand white rabits to prepare polyclonal antibody. Western blotting analyses showed that the generated anti-ANF polyconal antibody has high sensitivity and specificity.

关 键 词：ANF基因 融合蛋白 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]