斑马鱼ISL1蛋白多克隆抗体的制备  

The Expression,Purification of ISL1 Zerbrafish Protein and Preparation of Its Polyclonal Antibody

作　　者：任恋[1] 唐超[1] 刘宪楚[1] 李容[1] 戴悦[1] 彭佩莹[1] 莫小阳[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：《中南医学科学杂志》2013年第2期135-139,共5页Medical Science Journal of Central South China

基　　金：国家自然科学基金项目(31172044);湖南省杰出青年科学基金(10JJ1006);湖南省教育厅优秀青年科技计划项目(11B079)

摘　　要：目的为了进一步研究ISL1作为第二生心区心脏前体细胞的分子标志在心脏发育中的功能,需要获得ISL1蛋白并制备其抗体。方法根据已报道的ISL1基因序列,以斑马鱼mRNA为模板进行PCR扩增得到ISL1部分编码区序列,然后将其连接到pET-28a载体上获得原核表达载体。将重组质粒进行酶切测序鉴定,然后利用大肠杆菌E.coli BL21对该重组质粒进行表达。经过IPTG诱导,采用Ni-IDA凝胶柱亲和纯化,将纯化的his-ISL1融合蛋白免疫新西兰大白兔制备ISL1多克隆抗体,并用Western Blot检测抗体效价。结果获得了ISL1原核表达重组融合蛋白及高效价的特异性兔抗ISL1多克隆抗体。结论本实验为ISL1在斑马鱼心脏发育中的新功能的进一步研究奠定了基础。Objective ISL1 is a molecular marker of precursor cells in Second Heart Field. In order to further study the function of ISL1 in heart development, we need to get the ISL1 protein and it＇s antibody. Methods and Results According to the reported ISL1 gene sequences, using the zebrafish mRNA as the template for PCR amplification, we got part of the coding sequence of ISL1. after that,we connected the fragment to pET-28a vector to construct a prokaryotic expression vector and identified it with restriction enzyme digestion and sequencing identification, then using the E. coli BI21 to express the recombinant plasmid. In the end,after the IPTG induction and Ni - IDA affinity gel column purification,we have finally got the prokaryotic expression recombinant fusion protein of ISL1 and its polyclonal antibody of high potency and specificity against rabbit by using the purified fusion protein to immune the New Zealand white rabbit and testing the valence of antibody with Western Blot detection. Conclusion This research lays a solid foundation for further study on the function of ISL1.

关 键 词：ISL1 斑马鱼 融合蛋白 多克隆抗体 

分 类 号：Q7[生物学—分子生物学]