TEF-1δ基因真核细胞稳定表达系统的构建与鉴定

CONSTRUCTION AND IDENTIFICATION OF STABLE EXPRESSION SYSTEM OF KARYOCYTES WITH TEF-1δ GENE

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。

Purpose: To construct and identify the stable expression system of karyocytes with TEF δ gene. Methods: Two stable transfections of CHO and COS7 cells with plasmid (pcDNA3.1/V5 His TOPO Vector) expressing TEF 1δ cDNA were established by using calcium phosphate and G418 selection protocols. Results: The results showed that, after G418 selection and western blotting analysis, 3 out of 10 CHO cell lines transfected with TEF 1δ cDNA expressed very high levels of TEF 1δ encoded protein with an approximately molecular weight of 31 kDa. as compared with vector control transfectants that showed no expression, and compared with the other cell lines that expressed relatively low proteins. Similarly, 4 out of 4 COS7 cell lines had significant overexpression of TEF 1δ encoded protein. The names of these stable transfection cell lines were CHO pcDNA3.1 TEF 1δ, #3, #6 and #14 as well as COS7 pcDNA3.1 TEF 1δ #4, #8, #14 and #17, respectively. Conclusion: These cell lines can be applied to functional studies of the TEF 1δ gene, and these are the optimal cell lines for studies on the underlying molecular carcinogenic mechanisms of Cd carcinogenesis.