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。HTH]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。
英文摘要:
Objective To construct eukaryotic expressing recombinant vector of canine transferrin receptor gene (TfR ), then to transfect Chinese hamster ovary (CHO) cells with the vector for establishment of stable expression of TfR in CHO cell line. Methods The full-length TfR fragment was amplified by RT-PCR from the canine cells (walter reed dog cell, WRD) and then inserted into eukaryotic expression vector pCDNA3. After identification with enzyme digestion and sequencing, the recombinant vector was transfected into CHO cells by TransLipid Transfection Reagent. The stable transfected CHO cell line was then established by screening cultures with G418, and the expression of TfR was identified by RT-PCR, Western blot and immunofluorescence, respectively. Results The eukaryotic expression vector pCDNA3-TfR was constructed successfully by checking with enzyme digestion and sequencing, and the highly expressed canine TfR was observed in CHO cells transfected with pCDNA3-TfR by using RT-PCR, Western blot and immunofluorescence, respectively. The stable CHO cell line with canine TfR expression was established. Conclusion The construction of the eukaryotic expression vector pCDNA3-TfR and the establishment of stable CHO cell line with TfR expression provide solid foundation for further experimental studies on the function of TfR.
