斑马鱼基因ATF4多克隆抗体的制备

Preparation of Polyclonal Antibody of ATF4 in Zebrafish

ATF4是含有bZIP结构域的ATF/CREB转录因子家族成员,对胚胎的发育以及细胞的增殖、分化有重要的调节作用。制备ATF4的多克隆抗体对于研究其在斑马鱼心脏发育过程中的作用有重要的意义。研究首先通过生物信息学方法,选择ATF4基因中特异性强、具亲水性的一段核苷酸序列(1 017 bp),通过PCR扩增,将片段重组到原核表达载体pET-28a,然后转化入Rosetta菌株中。经测序鉴定正确后,用IPTG诱导表达融合蛋白,以该融合蛋白免疫小鼠,获得ATF4多克隆抗鼠血清。对该多抗血清抗体进行验证,具有很好特异性和较高效价,可以用作Western-blotting、免疫印迹等试验分析。

ATF4, a member of the ATF/CREB transcription factor family which contains a bZIP domain, plays a important role in the embryonic development, cell proliferation and differentiation. Therefore, it is advisable to prepare an anti- body for the heart development research in Zerbrafish. In this research, a ATF4-specific, hydrophilic nucleotide sequence （about 1 017 bp） was selected by using bioinformatics methods, and the sequence was amplified by PCR, the PCR product was inserted into the prokaryotic expression vector pET-28a and then transformed into Rosetta cells. After the sequence result of the recombinant plasmid pET-28a-ATF4 is verified to be right, the fusion protein was induced to express in Rosetta cells by IPTG. The mouse was immunized by injecting appropriate amount of fusion protein, and the ATF4 polyclonal antibody was obtained. The antibody＇ s titer and specification was identified. Our results showed that the ATF4 polyclonal antibody was practical and available for western-blotting analysis and immunohisto-chemistry.