CAJ | 학술논문

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,Western blot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,Western blot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.
목적 구건간암상관단백FAM172A2적원핵표체재체,유도융합단백적표체,병대기진행순화；제비토항FAM172A2단백다극륭항체병진행감정.방법 응용역전록-취합매련반응(RT-PCR)기술,이HepG2세포총RNA위모판,확증FAM172A2목적기인편단1113bp,구건원핵표체재체,Western blot분석증실융합단백표체적특이성.유도기대량표체후순화、복성병제비다극륭항체,Western blot급매련면역흡부시험(ELISA)검측특이성급기효개.결과 확증획득FAM172A2기인편단,성공유도FAM172A2융합단백표체병제비기다극륭항체.ELISA검측증실기효개＞1∶160000,차특이성량호.결론 이용대장애희균BL21( DE3)능구성공표체FAM172A2단백,획득고특이성、고효개토항FAM172A2단백적다극륭항체.
Objective To express and purify of the FAM172A2 recombinant protein,and to prepare the FAM172A2 specific rabbit polyclonal antibody.Methods FAM172A2 cDNA (1113 bp) was obtained 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 polyclonal 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 antiFAM172A2 polyclonal antibody will be valuable tools for the investigation on the biological function of FAM172A2.