本文選題：轉(zhuǎn)鐵蛋白受體 + CHO細(xì)胞��； 參考：《四川大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2017年01期

【摘要】：目的構(gòu)建犬轉(zhuǎn)鐵蛋白受體(transferrin receptor,TfR)的真核表達(dá)載體,轉(zhuǎn)染中國(guó)倉(cāng)鼠卵巢CHO細(xì)胞,建立穩(wěn)定表達(dá)犬TfR的CHO-TfR細(xì)胞系。方法從犬腺細(xì)胞系(WRD)提取總RNA,逆轉(zhuǎn)錄合成cDNA,采用PCR方法擴(kuò)增TfR基因,將其克隆至真核表達(dá)載體pCDNA3,酶切和測(cè)序鑒定;利用脂質(zhì)體法轉(zhuǎn)染CHO細(xì)胞,通過(guò)RT-PCR、Western blot及免疫熒光法檢測(cè)TfR的表達(dá)。結(jié)果構(gòu)建的pCDNA3-TfR重組質(zhì)粒經(jīng)過(guò)酶切和測(cè)序鑒定,TfR基因序列成功插入到pCDNA3載體,且無(wú)突變。轉(zhuǎn)染CHO細(xì)胞后,RT-PCR、Western blot及免疫熒光法檢測(cè)TfR表達(dá)陽(yáng)性,成功建立了穩(wěn)定表達(dá)犬TfR的CHO-TfR細(xì)胞系。結(jié)論成功構(gòu)建了真核表達(dá)載體pCDNA3-TfR,并獲得了穩(wěn)定表達(dá)TfR的CHO-TfR細(xì)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(xué)生物化學(xué)與分子生物學(xué)系寧夏醫(yī)科大學(xué)生育力保持教育部重點(diǎn)實(shí)驗(yàn)室;寧夏醫(yī)科大學(xué)總醫(yī)院呼吸與危重醫(yī)學(xué)科;
【基金】：國(guó)家自然科學(xué)基金(No.81260247)資助
【分類號(hào)】：R392

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本文編號(hào)：1998705