myh6蛋白的表达、纯化及多克隆抗体制备

Expression,Purification of myh6 Fusion Protein and Preparation of Its Polyclonal Antibody

斑马鱼心脏发育模型中,myh6编码是一种促进心室心肌细胞扩张的转录因子。为了进一步研究myh6在心脏发育和心脏疾病发生中的功能,需要获得斑马鱼myh6蛋白并制备其抗体。从斑马鱼心脏组织中提收总RNA,通过反转录得到心脏组织特异表达基因的cDNA,PCR扩增得到myh6部分编码区序列,然后将其连接到pGEX-4T载体上获得原核表达。经酶切及测序鉴定后,转化BL21细菌,并用IPTG诱导表达融合蛋白,谷胱甘肽琼脂糖珠亲和纯化,将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western blotting检测抗体的特异性。结果显示,获得了myh6原核表达重组融合蛋白及高效价的特异性兔抗myh6多克隆抗体,为myh6功能的进一步研究提供了有力的工具。

In Zebrafish heart development model, myh6 encodes transcription factors which can promote expansion of ventricular myocardial cells. In order to study the function of myh6 in heart development and heart disease, it is necessa- ry to obtian Zebrafish myh6 protein and myh6 antibody. The total RNA of Zebrafish heart tissues was extracted and was reverse transcrripted into cDNA as template by inverse transcriptional PCR. myh6 partial coding sequence was obtained by PCR amplification, then was cloned into pGEX-4T-1 vector and was identified with restriction enzyme digestion and sequencing. The recombinant expression plasmid containing myh6 gene was transformed into BL21 and GST-myh6 fusion protein was induced by IPTG. The purified protein obtained by treating the lysates with immobilized Glutathione Sepha- rose and then was used to immunize the New Zealand white rabbits to generate antibody. The antibody titer and specifici- ty was identified by Western blot. All these results showed that GST-myh6 fusion protein was purified successfully and the high sensitivity and high specificity anti-myh6 polyclonal antibody was generated, which provided a powerful tool for the further studies of myh6 function.