乙酰胆碱受体四聚体前体α-亚单位在HEK293细胞膜上的表达

发布时间：2018-06-06 16:26

  本文选题：乙酞胆碱受体 + HEK293细胞　； 参考：《延边大学》2011年硕士论文

【摘要】：目的：乙酰胆碱受体四聚体前体α亚单位(pcDNA3.1-AchRa-BirA)重组质粒转染至人胚肾细胞(HEK293)并在其细胞膜上获得稳定表达。为构建乙酰胆碱受体四聚体并将其作为抗原用于检测乙酰胆碱受体抗体的后续研究奠定基础。 方法：构建重组质粒载体pcDNA3.1-AchRa-BirA,将其转化至感受态细胞E. coli DH5a中。扩增培养E. coli DH5a,提取重组质粒pcDNA3.1-AchRa-BirA,经限制性核酸内切酶EcoRV单酶切后,采用低熔点琼脂糖凝胶电泳检测,初步确定提取到质粒。设计引物,经生物公司合成并测序,验证其基因序列。将成功提取到的重组质粒pcDN A3.1-AchRa-BirA,采用脂质体转染法转染至HEK293细胞。经G418筛选后,形成集落状抗性克隆细胞。将表达产物扩大培养,应用免疫荧光技术,以异硫氰酸荧光素(FITC)标记,经荧光显微镜查看表达情况。 结果：电泳检查发现了大小约为6964bp的条带,并经生物公司测得其基因全序列。转染后,G418筛选获得抗性克隆细胞,经荧光显微镜观察到HEK293细胞膜上的绿色荧光。 结论：成功提取了pcDNA3.1-AchRa-BirA识别多肽序列基因重组载体。经荧光显微镜观察到HEK293细胞膜上的绿色荧光,证明AchRa-BirA基因成功转染至HEK293细胞膜上且有表达。
[Abstract]:Aim: to transfect the recombinant plasmid pcDNA3.1-AchRa-BirA into human embryonic kidney cell line HEK293) and to obtain stable expression on the cell membrane of the recombinant plasmid pcDNA3.1-AchRa-BirA. To construct the tetramer of acetylcholine receptor and use it as antigen to detect the antibody of acetylcholine receptor. Methods: recombinant plasmid pcDNA3.1-AchRa-BirA was constructed and transformed into E. coli DH5a. The recombinant plasmid pcDNA3.1-AchRa-BirA was extracted by amplification and culture. The plasmid was identified by low melting point agarose gel electrophoresis after the restriction endonuclease EcoRV was digested. The primer was designed and synthesized and sequenced by a biological company to verify its gene sequence. The recombinant plasmid pcDN A3.1-AchRa-BirA was transfected into HEK293 cells by liposome transfection. Colony resistant clone cells were formed after G418 screening. The expression product was expanded in culture and labeled with fluorescein isothiocyanate by immunofluorescence technique. The expression was examined by fluorescence microscope. Results: the 6964bp bands were found by electrophoresis, and the whole gene sequence was obtained by biological company. After transfection, the resistant clone cells were screened by G418, and the green fluorescence on HEK293 cell membrane was observed by fluorescence microscope. Conclusion: pcDNA3.1-AchRa-BirA recognition polypeptide gene recombinant vector was successfully extracted. The green fluorescence on HEK293 cell membrane was observed by fluorescence microscope, which proved that AchRa-BirA gene was successfully transfected into HEK293 cell membrane and expressed.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【参考文献】

中国期刊全文数据库 前2条

1 唐吟岫,谈华,虞伟,武建国;斑点免疫结合实验用于乙酰胆碱受体抗体的检测[J];医学研究生学报;2000年06期

2 龚其美,李婉宜,李明远,李毓琦,牟家琬,徐文帧;快速检测重症肌无力患者的抗乙酰胆碱受体抗体的研究[J];临床检验杂志;2002年01期

，

本文编号：1987371