小鼠甲胎蛋白基因的克隆表达鉴定及稳定表达甲胎蛋白的小鼠肿瘤细胞株的建立

Gene cloning and expression identification of murine a-fetoprotein gene and establishment of a cell line stably expressing α-fetoprotein

目的:克隆小鼠甲胎蛋白(AFP)基因并进行表达鉴定,构建小鼠AFP真核表达载体并建立稳定表达甲胎蛋白的小鼠肿瘤细胞株．方法:从HePal-6细胞中提取总RNA进行 RT-PCR,克隆出小鼠AFP基因,亚克隆于 pcDNA3．1中构建真核表达载体pmAFP,进行酶切、测序和表达鉴定,pmAFP稳定转染 EL-4细胞建立EL-4(mAFP)细胞株,RT-PCR 检测EL-4(mAFP)细胞mAFP mRNA的表达,Western blot检测mAFP蛋白表达,将EL- 4(mAFP)N胞种植于6只小鼠背部皮下观察成瘤情况．结果:RT-PCR成功克隆出小鼠AFP基因,酶切、测序和表达鉴定证实真核表达载体 pmAFP构建成功,RT-PCR及Western blot检测证实EL-4(mAFP)细胞中有mAFP mRNA及蛋白的表达,5只小鼠背部皮下种植处有肿瘤形成,22 d肿瘤体积为2 279．97±235．13 mm3．结论:小鼠mAFP基因克隆成功,真核表达载体构建成功,建立的EL-4(mAFP)细胞株能有效表达小鼠甲胎蛋白,在小鼠体内成瘤性良好．

AIM： To clone the murine α-fetoprotein （mAFP） gene, construct the eukaryotic expression vector of AFP and establish a cell line stably expressing AFP. METHODS： The total RNA was extracted from Hepal-6 cells. The mAFP gene was amplified by reverse transcription polymerase chain reaction （RT-PCR） and cloned into the eukaryotic expression vector pcDNA3.1 to construct pmAFP. The pmAFP was identified by restriction enzyme analysis and sequencing, and then stably transfected into EL-4 cell line. Western blot and RTPCR were used to detect the expression of mAFP protein and mRNA, respectively. EL-4 cells stably expressing mAFP were inoculated in the back of 6 mice to observe the tumor formation. RESULTS： The mAFP gene with a length of 1.8 kb was successfully cloned from the total RNA of Hepal-6 cells. Restriction enzyme analysis and sequencing showed that the 1.8 kb mAFP gene was successfully cloned and inserted into pcDNA3.1. RT-PCR and Western blot showed rnAFP was stably expressed in EL-4 cell line. The tumor grew to a volume of 2 279.97 ± 235.13 mm^3 22 d after inoculation in 5 mice. CONCLUSION： The InAFP gene is successfully cloned and a cell line stably expressing mAFP, named EL-4 （mAFP）, is established. EL4 （mAFP） has a good tumor-forming capacity in mice.