MRF4 is one of muscle regulatory factors and plays critical roles during skeletal muscle development. The muscle development is important for the fish growth which is an important economic factor for the fish culture. To analyze the function of MRF4 in fish, the founder MRF4 antibody was prepared. The flounder MRF4 was cloned, ligated into prokaryotic expression vector pET-30b and expressed in strain E. coli BL21 (DE3). The recombinant flounder MRF4 fusion protein was soluble and purified with cobalt IMAC resins. To prepare MRF4 polyclonal antibodies, rabbits were immunized with the soluble protein and the increasing level of antibodies was determined by Western blot. Also, the endogenous flounder MRF4 was recognized by the anti-serum. The result further proved the existence of the anti-MRF4 antibody in the anti-serum, which will be useful for studies on the function of flounder MRF4.%MRF4是肌肉发生调节因子家族的一员,其对肌肉的发育有重要的调节作用.对于养殖鱼类,生长速度是很重要的经济指标,而肌肉的发育对鱼类的生长非常重要.制备了MRF4的多克隆抗体以分析在鱼类中MRF4的功能.通过采用大肠杆菌表达系统及pET-30表达载体对牙鲆肌肉发生调节因子MRF4进行了体外重组表达.所得到的MRF4重组蛋白为可溶性的.在自然条件下,经金属离子螯合柱纯化后,得到了电泳纯的融合蛋白,通过免疫新西兰大白兔制备MRF4的多克隆抗体.蛋白质印迹分析显示:重组蛋白能被抗组氨酸标签的抗体及所制备的多克隆抗体识别,而且这一多克隆抗体能检测到内源性的MRF4蛋白,从而进一步证明了抗血清中含有MRF4的抗体.MRF4融合蛋白的成功表达及其多克隆抗体的制备为进一步深入研究其在牙鲆肌肉发育过程中的分子机制奠定了基础.
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