重组乙酰胆碱受体α亚单位-BirA识别多肽序列基因的构建与表达

Construction and expression of recombinant acetylcholine receptor α subunit-BirA recognition polypeptide sequence gene

[目的]构建重组乙酰胆碱受体亚单位-BirA识别多肽序列基因(pcDNA 3.1-AChR-αBirA),并对其进行真核基因表达.[方法]设计引物生物素蛋白连接酶(BirA)识别多肽序列基因,将载体pcDNA3.1-AChR-αBirA识别多肽序列基因连接,并用EcoRⅤ单酶切检查BirA识别多肽序列基因插入的正确性,并测定序列进行验证.将构建成功的载体在中国仓鼠卵巢(CHO)细胞上进行表达,经激光扫描共聚焦显微镜验证其产物.[结果]电泳检查可见大小为63 bp的条带,经测定序列验证了插入的基因序列为BirA识别多肽序列基因;转染后经激光扫描共聚焦显微镜观察到CHO细胞膜上的绿色荧光.[结论]成功构建了AChR-αBirA识别多肽序列基因重组载体,并可在CHO细胞中较好地表达.

OBJECTIVE To construct recombinant acetylcholine receptor a subunit-BirA recognition polypeptide sequence gene （pcDNA 3.1-AChRa-BirA） and to express it in eukaryotic cell line. METHODS The biotin-protein ligase （BirA） recognition polypeptide sequence gene was designed and synthesized. The eukaryotic expression vector pcDNA 3.1 bearing human AChR αsubunit （pcDNA 3.1-AChRa was ligated with the synthesized BirA recognition polypeptide sequence gene. The recombinant vector pcDNA 3.1-AChRα-BirA recognition polypeptide sequence gene was digested with EcoRⅤ. The pcDNA 3.1- AChRa-BirA recognition polypeptide sequence gene was analysed for an insert of the right size by digestion with EcoRⅤ and verified by sequencing method.The pcDNA 3.1-AChRα-BirA recognition polypeptide sequence was subsequently expressed in eukaryotic CHO cells. Green fluorescence staining labeled expression products were verified by confocal laser scanning microscopy. RESULTS The band corresponding to the BirA recognition polypeptide sequence gene was 63 bp in size as revealed in electrophoretic examination, and the sequence was correct as verified by sequencing. The green fluorescence could be seen on the cell membrane as observed by confocal laser scanning microscopy after transfection. CONCLUSIONS The recombinant vector pcDNA 3.1-AChRα-BirA recognition polypeptide sequence gene is successfully constructed, and expressed in CHO cells. This experiment lays the foundation for constructing tetrameric AChRα to be used as the antigen in the determination of anti-AChR antibodies.