稳定表达犬转铁蛋白受体CHO细胞系的建立和鉴定

发布时间：2018-06-09 03:38

本文选题：转铁蛋白受体 + CHO细胞　；参考：《四川大学学报(医学版)》2017年01期

【摘要】：目的构建犬转铁蛋白受体(transferrin receptor,TfR)的真核表达载体,转染中国仓鼠卵巢CHO细胞,建立稳定表达犬TfR的CHO-TfR细胞系。方法从犬腺细胞系(WRD)提取总RNA,逆转录合成cDNA,采用PCR方法扩增TfR基因,将其克隆至真核表达载体pCDNA3,酶切和测序鉴定;利用脂质体法转染CHO细胞,通过RT-PCR、Western blot及免疫荧光法检测TfR的表达。结果构建的pCDNA3-TfR重组质粒经过酶切和测序鉴定,TfR基因序列成功插入到pCDNA3载体,且无突变。转染CHO细胞后,RT-PCR、Western blot及免疫荧光法检测TfR表达阳性,成功建立了稳定表达犬TfR的CHO-TfR细胞系。结论成功构建了真核表达载体pCDNA3-TfR,并获得了稳定表达TfR的CHO-TfR细胞系。
[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.
【作者单位】：宁夏医科大学生物化学与分子生物学系宁夏医科大学生育力保持教育部重点实验室;宁夏医科大学总医院呼吸与危重医学科;
【基金】：国家自然科学基金(No.81260247)资助
【分类号】：R392

【参考文献】