小鼠骨形成蛋白BMP3的原核表达及多克隆抗体制备与鉴定

Prokaryotic Expression,Polyclonal Antibody Preparation and Identification of Mouse BMP3 Protein

为构建小鼠BMP3蛋白原核表达载体,纯化BMP3蛋白,制备并鉴定BMP3蛋白小鼠多克隆抗体,以及对BMP3进行系统进化关系研究。采用RT-PCR方法从小鼠肝脏中克隆获得BMP3蛋白的表达菌株;利用SDS-PAGE电泳切胶纯化、尿素梯度复性获得BMP3蛋白,免疫小鼠制备BMP3蛋白多克隆抗体;采用ELISA法检测抗体滴度达1∶3 200倍,Western Blotting证实抗血清具有很好的特异性。通过DNAMAN软件对BMP3序列同源性分析发现其C-端在不同动物间存在较高同源性;MEGA软件对BMP3序列的系统发生分析证实BMP3在动物间具有明显的进化趋势。为BMP3蛋白调控骨代谢及与肿瘤的关系研究奠定基础。

To construct prokaryotic expression vector of mouse bone morphogenic protein 3（ BMP3）,prepare the polyclonal antibodies of mouse anti-BMP3,and make a study on the phylogenetic relationships of BMP3 protein.The BMP3 expression strain was obtained from mouse liver by RT-PCR and molecular clone; BMP3 was purified by the way of SDS-PAGE gel slices,renatured by urea gradient,and immunized mice to prepare the polyclonal antibody. BMP3 antibody titer was 1 ∶ 3 200 by ELISA; and Western Blotting proved that the antiserum had good specificity.BMP3 homology analysis found that the C-terminal had high homology in different animal by DNAMAN software;BMP3 sequence phylogenetic analysis showed that the animal evolution trend was different by MEGA software.These works laid the groundwork for the function of BMP3 regulated bone metabolism and the relationship between BMP3 and tumor.