本文選題：轉鐵蛋白受體 + CHO細胞��； 參考：《四川大學學報(醫(yī)學版)》2017年01期

【摘要】：目的構建犬轉鐵蛋白受體(transferrin receptor,TfR)的真核表達載體,轉染中國倉鼠卵巢CHO細胞,建立穩(wěn)定表達犬TfR的CHO-TfR細胞系。方法從犬腺細胞系(WRD)提取總RNA,逆轉錄合成cDNA,采用PCR方法擴增TfR基因,將其克隆至真核表達載體pCDNA3,酶切和測序鑒定;利用脂質體法轉染CHO細胞,通過RT-PCR、Western blot及免疫熒光法檢測TfR的表達。結果構建的pCDNA3-TfR重組質粒經(jīng)過酶切和測序鑒定,TfR基因序列成功插入到pCDNA3載體,且無突變。轉染CHO細胞后,RT-PCR、Western blot及免疫熒光法檢測TfR表達陽性,成功建立了穩(wěn)定表達犬TfR的CHO-TfR細胞系。結論成功構建了真核表達載體pCDNA3-TfR,并獲得了穩(wěn)定表達TfR的CHO-TfR細胞系。
[Abstract]:Objective to construct the eukaryotic expression vector of dog transferrin receptor TfR and transfect it into Chinese hamster ovary Cho cells, and to establish a stable CHO-TfR cell line expressing dog TfR. Methods Total RNAs were extracted from canine adenoma cell line WRDand cDNAs were synthesized by reverse transcription. TfR gene was amplified by PCR, cloned into eukaryotic expression vector pCDNA3, digested and sequenced, and transfected into Cho cells by liposome method. The expression of TFR was detected by RT PCR Western blot and immunofluorescence assay. Results the recombinant plasmid pCDNA3-TfR was successfully inserted into pCDNA3 vector by restriction endonuclease digestion and sequencing. After transfection of Cho cells, RT-PCR blot and immunofluorescence assay were used to detect the expression of TFR, and a stable CHO-TfR cell line expressing dog TfR was successfully established. Conclusion the eukaryotic expression vector pCDNA3-TfR was successfully constructed and the CHO-TfR cell line expressing TfR stably was obtained.
【作者單位】： 寧夏醫(yī)科大學生物化學與分子生物學系寧夏醫(yī)科大學生育力保持教育部重點實驗室;寧夏醫(yī)科大學總醫(yī)院呼吸與危重醫(yī)學科;
【基金】：國家自然科學基金(No.81260247)資助
【分類號】：R392

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本文編號：1998705