摘要
小鼠MDFIC(MyoD family inhibitor domain containing)蛋白是一个新近发现的与HIC(Human I-mfa domain containing protein)高度同源的转录调控因子,可能在肌细胞的分化过程中起着重要的作用。为了深入研究MDFIC的功能,首先要制备抗MDFIC的多克隆抗体。首先采用PCR方法扩增出编码MDFIC的cDNA片段,然后将其克隆至原核表达载体pGEX-4T-3,并在大肠杆菌BL21中进行诱导表达。SDS-PAGE和Western blotting检测鉴定表达产物。亲和层析技术对融合蛋白进行纯化,纯化后的目的蛋白免疫新西兰兔制备多克隆抗体。间接ELISA检测抗体效价达1∶640 000以上,检测证明抗体效价较高。此外,Western blotting结果显示,该抗体可特异性识别真核细胞外源性及内源性的MDFIC蛋白。MDFIC多克隆抗体的成功制备为进一步研究MDFIC的功能以及进一步探索肌细胞的发生分化机制提供了重要工具。
Mouse MDFIC（MyoD family inhibitor domain containing） protein is a newly found transcriptional factor,which shows high homology with HIC（Human I-mfa domain containing protein） and may play important roles in myogenic differentiation.cDNA encoding full-length of MDFIC was amplified by PCR and was inserted into prokaryotic expression vector pGEX-4T-3.The recombinant plasmid pGEX-4T-3-MDFIC was then transformed into E.coli BL21 and induced.The expression product was indentified by SDS-PAGE and Western blotting analysis.The fusion protein was purified by affinity chromatography with glutathione sepharose-4B column and then was used to immunize rabbits to generate polyclonal antibody.Indirect ELISA analysis shows that the titer of the antibody is higher than 1∶640 000.Besides,Western blotting analysis indicates that the antibody can detect both exogenous and endogenous MDFIC protein.The polyclonal antibody of MDFIC provides an important tool to study the function of MDFIC and further to explore the mechanisms of myogenesis.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2010年第2期49-53,共5页
China Biotechnology
基金
高等学校博士学科点专项科研基金资助项目(20060559006)
关键词
MDFIC
转录因子
多克隆抗体
肌细胞分化
MDFIC Transcriptional factor Polyclonal antibody Myogenic differentiation
