本文選題：乙酞膽堿受體 + HEK293細胞　； 參考：《延邊大學》2011年碩士論文

【摘要】：目的：乙酰膽堿受體四聚體前體α亞單位(pcDNA3.1-AchRa-BirA)重組質(zhì)粒轉(zhuǎn)染至人胚腎細胞(HEK293)并在其細胞膜上獲得穩(wěn)定表達。為構建乙酰膽堿受體四聚體并將其作為抗原用于檢測乙酰膽堿受體抗體的后續(xù)研究奠定基礎。 方法：構建重組質(zhì)粒載體pcDNA3.1-AchRa-BirA,將其轉(zhuǎn)化至感受態(tài)細胞E. coli DH5a中。擴增培養(yǎng)E. coli DH5a,提取重組質(zhì)粒pcDNA3.1-AchRa-BirA,經(jīng)限制性核酸內(nèi)切酶EcoRV單酶切后,采用低熔點瓊脂糖凝膠電泳檢測,初步確定提取到質(zhì)粒。設計引物,經(jīng)生物公司合成并測序,驗證其基因序列。將成功提取到的重組質(zhì)粒pcDN A3.1-AchRa-BirA,采用脂質(zhì)體轉(zhuǎn)染法轉(zhuǎn)染至HEK293細胞。經(jīng)G418篩選后,形成集落狀抗性克隆細胞。將表達產(chǎn)物擴大培養(yǎng),應用免疫熒光技術,以異硫氰酸熒光素(FITC)標記,經(jīng)熒光顯微鏡查看表達情況。 結果：電泳檢查發(fā)現(xiàn)了大小約為6964bp的條帶,并經(jīng)生物公司測得其基因全序列。轉(zhuǎn)染后,G418篩選獲得抗性克隆細胞,經(jīng)熒光顯微鏡觀察到HEK293細胞膜上的綠色熒光。 結論：成功提取了pcDNA3.1-AchRa-BirA識別多肽序列基因重組載體。經(jīng)熒光顯微鏡觀察到HEK293細胞膜上的綠色熒光,證明AchRa-BirA基因成功轉(zhuǎn)染至HEK293細胞膜上且有表達。
[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.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

【參考文獻】

中國期刊全文數(shù)據(jù)庫 前2條

1 唐吟岫,談華,虞偉,武建國;斑點免疫結合實驗用于乙酰膽堿受體抗體的檢測[J];醫(yī)學研究生學報;2000年06期

2 龔其美,李婉宜,李明遠,李毓琦,牟家琬,徐文幀;快速檢測重癥肌無力患者的抗乙酰膽堿受體抗體的研究[J];臨床檢驗雜志;2002年01期

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本文編號：1987371