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[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 pcDNA3.1-AChR-αBirA, a recombinant subunit of acetylcholine receptor, and express it in eukaryotic gene. [Methods] Primer biotin ligase (BirA) Gene and the vector pcDNA3.1-AChR-αBirA recognition polypeptide gene was ligated, and the correctness of insertion of BirA recognition polypeptide sequence was verified by EcoRⅤ digestion and the sequence was verified. The constructed vector was successfully transfected into Chinese hamster ovary ). The product was confirmed by laser scanning confocal microscopy. [Results] A 63 bp band was observed by electrophoresis, and the inserted sequence was verified to be BirA recognition polypeptide sequence. After transfection The green fluorescence of CHO cell membrane was observed by laser scanning confocal microscope. [Conclusion] The AChR-αBirA recognition polypeptide sequence gene recombinant vector was successfully constructed and expressed well in CHO cells.