摘要
目的构建肝癌相关蛋白FAM172A2的原核表达载体，诱导融合蛋白的表达，并对其进行纯化；制备兔抗FAM172A2蛋白多克隆抗体并进行鉴定。方法应用逆转录．聚合酶链反应（RT-PCR）技术，以HepG2细胞总RNA为模板，扩增FAM172A2目的基因片段1113bp，构建原核表达载体，Westernblot分析证实融合蛋白表达的特异性。诱导其大量表达后纯化、复性并制备多克隆抗体，Westernblot及酶联免疫吸附试验（ELISA）检测特异性及其效价。结果扩增获得FAM172A2基因片段，成功诱导FAM172A2融合蛋白表达并制备其多克隆抗体。ELISA检测证实其效价〉1：160000，且特异性良好。结论利用大肠埃希菌BL21（DE3）能够成功表达FAM172A2蛋白，获得高特异性、高效价兔抗FAM172A2蛋白的多克隆抗体。
Objective To express and purify of the FAM172A2 recombinant protein, and to pre- pare the FAM172A2 specific rabbit polyclonal antibody. Methods FAM172A2 eDNA （1113 bp） was ob- tained by reverse transcription-polymerase chain reaction （RT-PCR）. The prokaryotic expressive vector was constructed. The protein expression was induced and the protein was analyzed with Western blotting. The purified pET-32a（ ＋ ）-FAM172A2 fusion protein was used to gain polyelonal antibody. The specificity and potency of polyclonal antibody were evaluated by Western blotting and enzyme linked immunosorbent assay （ELISA）. Results The FAM172A2 fusion protein was highly expressed. The protein was mainly in the inclusion body. ELISA indicated the titer of polyclonal antibody 〉 1 ： 160 000. The high specificity was confirmed with Western blotting. Conclusion The recombinant FAM172A2 fusion protein and the anti- FAM172A2 polyclonal antibody will be valuable tools for the investigation on the biological function of FAM172A2.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第3期432-434,共3页
Chinese Journal of Experimental Surgery
关键词
原核表达
蛋白纯化
Prokaryotic expression
Protein purification
