重组人乙酰胆碱受体α亚单位胞外域在CHO细胞中的表达

发布时间：2018-05-17 20:35

本文选题：重症肌无力 + 乙酰胆碱受体　；参考：《细胞与分子免疫学杂志》2013年02期

【摘要】：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。
[Abstract]:Objective to obtain recombinant human nicotinic acetylcholine receptor (nAChR) 伪 1 extracellular domain (ECD) protein with biological activity. Methods the full-length gene of human nAChR 伪 1 subunit was extracted from TE761 cells. The nAChR 伪 1 subunit ECD1-216 was amplified by PCR. The gene was digested into pcDNA3.1 expression vector and transfected into CHO cells by liposome. The expression level of ECD gene was detected by fluorescence real-time PCR at 7 time points at 6h 72 h after transfection, and the expression of ECD protein in the supernatant of cell culture was detected by 125I-labeled Agkistrotoxin. Results the size of the 708bp fragment obtained after H 伪 1-216 PCR was in accordance with the predicted results, and the nucleotide sequence obtained by sequencing was completely consistent with the human AChR 伪 1 sequence in GenBank. The target gene was inserted correctly and the vector was successfully constructed. With the extension of cell culture time, the level of mRNA expression of ECD gene increased gradually, and the expression of ECD protein increased. Conclusion Recombinant human nAChR 伪 1 extracellular domain protein with biological activity was successfully expressed in CHO cells.
【作者单位】：第四军医大学唐都医院神经内科;第四军医大学病原与微生物学教研室;
【基金】：国家自然科学基金(30972721)
【分类号】：R392.12

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