斑马鱼CFL基因的克隆、多克隆抗体的制备及分析

Prokaryotic expression and purification of the zebrafish CFL gene and preparation of its polyclonal antibody

在心脏发育过程中,相关基因的正常表达是有功能心脏形成的关键.斑马鱼CFL基因是从斑马鱼胚胎的cDNA文库中克隆出来的一个心脏发育候选基因.生物信息学分析表明:CFL基因编码278个氨基酸,含有一个CXXC结构域.为进一步研究该基因的功能,采用生物信息学结合RT-PCR的方法获得了该基因.将所得片段插入原核表达载体pET-28a载体中,经测序鉴定正确后转化入Rosetta菌株中,用IPTG诱导表达出pET-28a-CFL融合蛋白,表达的融合蛋白占菌体总蛋白的71%,融合蛋白相对分子质量约为30 kD.经包涵体纯化后,免疫新西兰大白兔制备了多克隆抗体.经验证,具有较好的特异性和较高的效价,可以用作western-blot免疫印迹等实验分析.

The normal expression of related genes is critical in the heart development process. To explore the heart development related genes, to provide the basis to understand the pathogenesis of congenital heart disease and interventional treatment. CFL gene is a candidate gene for heart development from Zebrafish cDNA library. Bioinformatics analysis revealed that it contains CXXC domain, and encoding 278 amino acids. To further investigate the possible functions of CFL in the process of heart development, the full length of CFL gene was cloned and inserted into pET-28a vector.The His-CFL fusion protein was induced by IPTG, the expression of fusion protein accounts for 71 of total bacteria protein. The relative molecular mass is about 30 kD. After inclusion body purification, immune the New Zealand white rabbit to prepared polyclonal antibody and was indentifited by western blot. These results demonstrate that the high titer polyclonal antibody was obtained.