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目的 :构建TEF 1δ (mousetranslationelongationfactor 1δ)的稳定表达系统。方法 :采用磷酸钙介导转染技术和G4 18细胞筛选法 ,以 pcDNA3.1/V5 His TOPO为表达载体 ,构建了TEF 1δ转基因CHO和COS7细胞系 ,WesternBlot分析与鉴定表达蛋白。结果 :在 10株转染和经G4 18反复筛选的CHO细胞系中 ,有 3株 (编码为COH pcDNA3.1 TEF 1δ # 3,# 6 ,# 14 )具有高效稳定表达的TEF 1δ编码蛋白质 (Mr约 31× 10 3 ) ,其余CHO细胞株的TEF 1δ蛋白质表达相对较弱或无表达。在 4株转染和经G4 18反复筛选的COS7细胞系中 ,4株细胞 (编码为COS7 pcCDNA3.1 TEF 1δ # 4 ,# 8,# 14和 # 17)均有高效稳定的TEF 1δ编码蛋白表达 ,相应的无转染组及载体对照组的CHO和COS7细胞均无TEF 1δ蛋白质表达。结论 :这两类TEF 1δ转基因哺乳动物细胞稳定表达系统已成功构建与鉴定 ,该表达系统的建立对于TEF 1δ这一新基因的生物学功能研究 ,尤其是镉的致癌作用与致癌机制的研究有重要应用价值。
Objective : To construct a stable expression system for TEF 1δ (mouse translation persistence factor 1δ). METHODS: Calcium phosphate-mediated transfection technique and G4 18 cell selection method were used. The pcDNA3.1/V5 His TOPO expression vector was used to construct TEF 1δ transgenic CHO and COS7 cell lines. Western blot was used to analyze and identify the expressed proteins. RESULTS: Among 10 strains of CHO cell lines transfected and repeatedly screened by G418, 3 strains (encoded as COH pcDNA3.1 TEF 1 δ #3, #6, #14) had highly efficient and stable expression of TEF 1δ-encoded proteins ( Mr was about 31×10 3 ). The expression of TEF 1δ protein in the other CHO cell lines was relatively weak or not. Among the 4 COS7 cell lines transfected and repeatedly screened by G418, 4 cells (encoded as COS7 pcCDNA3.1, TEF1δ#4, #8, #14 and #17) had highly efficient and stable TEF1δ encoded protein. Expression, the corresponding non-transfected group and vector control group CHO and COS7 cells have no TEF 1δ protein expression. Conclusion: These two types of TEF 1δ transgenic mammalian cell stably expressing system have been successfully constructed and identified. The establishment of this expression system has been used to study the biological function of TEF 1δ, especially the carcinogenic effect and carcinogenic mechanism of cadmium. Important application value.