表达肾癌G250基因和hGM-CSF基因HEK293细胞系的建立

目的 建立稳定表达肾癌G250基因与基因佐剂hGM-CSF基因编码蛋白的的HEK293细胞系。方法 通过脂质体转染的方法，将表达肾癌G250基因与细胞因子GM-CSF基因的双顺反子pIG250-GM真核表达质粒导入HEK293细胞中；经持续G418压力选择和有限稀释法获得稳定转染的细胞系；用免疫组化、ELISA和Western Blot方法，检测目的蛋白在HEK293细胞中的表达。结果 经600 μg/mL的G418压力筛选后，获得了抗性细胞克隆；免疫组织化学方法检测到表达G250的阳性细胞；ELISA方法检测到pIG250-GM转染组细胞培养上清中hGM-CSF的表达与空载体对照组比较，差异有统计学意义（P<0.05）；Western Blotting方法检测到G250蛋白的表达，分子量约54Ku，与预期大小相符。结论 成功建立可稳定表达肾癌G250基因与细胞因子GM-CSF基因的HEK293细胞系，为研究防治肾癌疫苗的免疫应答机制提供了实验基础。

Construction of HEK293 cell line containing kidney cancer G250 genes and hGM-CSF genes

Objective To construct a stable expression HEK293 cell line,.which containing G250 genes of renal carcinoma and gene adjuvant hGM-CSF. Methods The recombinant plasmid pIG250-GM containing G250 gene and hGM-CSF gene was transfected into the HEK293 cells by liposome..After selected by resistance to G418 and isolated with a limited dilution,.the stable transfected cell line was obtained..At the end,we detected the expression of tG250 and hGM-CSF protein by immunocytochemistry,.ELISA and Western blot. Results T...