人肌纤生成调节因子-1抗体的制备、鉴定及其在乳鼠心肌细胞中的应用  

Preparation and characterization of the antibodies against human myofibrillogenesis regulator 1 and application in neonatal rat cardiomyocytes

作　　者：王晓礽[1] 刘秀华[1] 宋泉声[2] 李婷[2] 韩文玲[2] 

机构地区：[1]解放军总医院病理生理研究室,北京100853 [2]北京大学人类疾病基因研究中心,北京100083

出　　处：《中国病理生理杂志》2010年第1期42-47,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30770902);国家自然科学基金重大国际合作项目资助(No.30620130111)

摘　　要：目的:制备抗人肌纤生成调节因子-1(hMR-1)的多肽抗体,并对其纯度、特异性、效价及适用性进行检测和鉴定。方法:TMHMM、DNAstar等软件分析hMR-1蛋白序列,选取2段优势抗原序列合成多肽并与钥孔戚血蓝素(KLH)偶联,混合后免疫家兔。抗血清经过免疫亲和层析法纯化得到多克隆抗体。用ELISA法检测效价,Western blotting和细胞免疫荧光(immunocytofluorescent,ICF)法鉴定其特异性和适用范围,并观察其在乳鼠心肌细胞中的应用情况。结果:(1)抗体效价达1∶105,Western blotting检测到与预测分子量17kD相符的条带,ICF图像背景低,阳性结构清晰。(2)乳大鼠心肌细胞实验发现荧光信号浓集于核周,过表达hMR-1后荧光强度明显高于空载对照和正常对照。结论:制备的抗hMR-1多肽抗体效价及特异性可用于Western blotting及ICF实验,可识别人源性和大鼠源性抗原表位,该工作将为进一步深入研究新基因hMR-1的功能奠定基础。AIM： To prepare and purify the polyclonal antibodies against human myofibrillogenesis regulator 1 （ hMR - 1 ）, then to characterize the purity, titer, specificity and the availability. METHODS ： Two polypeptides named peptide 1 and 2 were synthesized based on the bioinformatics analysis of the sequence of hMR - 1 by using software TMHMM and DNAStar, then coupled with keyhole limpet hemocyanin （KLH） for immunization. These peptides for immunization were mixed and injected into New Zealand rabbits to prepare antibodies specifically against hMR - 1. ELISA assay was used to detect the titers of the antibodies. After purification by immunoaffinity chromatography, antibodies were identified by Western blotting and immunocytofluorescent assays. Applications of the antibodies on neonatal rat cardiomyocytes were also employed. RESULTS： （1）The titers of antibodies were 1：105. In WB assay, a specific 17kD band was detected, corresponding to the predicted molecular weight of hMR - 1 ; the positive fluorescent signals were distinct. （2） On the neonatal rat cardiomyocytes model, we observed a peri - nucleus location. The fluorescent signal of hMR - 1 overexpression group was much stronger than that in vector control and normal control groups. CONCLUSION： All these results indicate that the antibodies obtained from poly peptides mixture immunization have either human original or rat original antigens. The antibody is available for using in Western blotting or immunofluorescent assays.

关 键 词：肌纤维生成调节因子1 抗体 乳鼠心肌细胞 

分 类 号：R541.4[医药卫生—心血管疾病] R363.2[医药卫生—内科学]