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目的获得具有生物学活性的重组人烟碱型乙酰胆碱受体(nAChR)α1亚基胞外域(ECD)蛋白。方法从TE761细胞中提取人nAChRα1亚单位全长基因,以PCR法扩增nAChRα1亚基ECD1-216,酶切后装入pcDNA3.1表达载体中,脂质体转染入CHO细胞,分别在转染后6～72 h的7个时间点进行荧光实时定量PCR鉴定ECD基因的表达水平,利用125I标记的银环蛇毒素测定细胞培养上清中ECD蛋白的表达。结果 Hα1-216 PCR后所得708 bp片段大小符合预计结果,测序所得核苷酸序列与GenBank中人AChRα1序列完全一致。所构建的新载体经酶切鉴定目的基因正确插入,载体构建成功。随着细胞培养时间的延长ECD基因mRNA表达水平逐渐增加,且ECD蛋白表达水平也不断提高。结论在CHO细胞成功表达了具有生物学活性的重组人nAChRα1胞外域蛋白。
Objective To obtain a biologically active recombinant human nicotinic acetylcholine receptor (nAChR) α1 subunit extracellular domain (ECD) protein. Methods The full-length human nAChRα1 subunit gene was extracted from TE761 cells. The nAChRα1 subunit ECD1-216 was amplified by PCR. The recombinant plasmid was subcloned into pcDNA3.1 expression vector and transfected into CHO cells. Real-time quantitative PCR was used to identify the expression of ECD gene at 7 time points from 6 to 72 h after staining, and the expression of ECD protein in cell culture supernatant was determined by 125I-labeled bungarotoxin. Results The size of the 708 bp fragment obtained after Hα1-216 PCR accorded with the expected result. The nucleotide sequence of the fragment was completely identical with the human AChRα1 sequence in GenBank. The constructed new vector was identified by restriction enzyme digestion of the correct insertion of the vector was successfully constructed. With the prolongation of cell culture time, the expression of ECD mRNA increased and the expression of ECD protein increased. Conclusion The recombinant human nAChRα1 extracellular domain protein with biological activity was successfully expressed in CHO cells.