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

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.