激光生物学报, 2011, 20 (5): 645, 网络出版: 2015-10-08
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
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321 bp, 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。
Abstract
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 prokaryotic 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.
李竞超, 吴坚, 梁艳, 莫小阳, 吴秀山. 斑马鱼ANF基因的克隆及多克隆抗体的制备[J]. 激光生物学报, 2011, 20(5): 645. LI Jingchao, WU Jian, LIANG Yan, MO Xiaoyang, WU Xiushan. Cloning and Polyclonal Antibody Preparation of ANF Gene in Zebrafish[J]. Acta Laser Biology Sinica, 2011, 20(5): 645.