摘要
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。
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 The stable transfected cell line was obtained after selected by G418 of 600 μg/mL.The expression of G250 positive cells was detected by immunocytochemistry,.and hGM-CSF in positive group was found by ELISA compared with empty vector control group, the difference was statistically significant（P0.05）. The G250 protein was detected by Western blot, the gene fragments were 54 Ku,which was consistent with the expectation. Conclusion The successful construction of a stable tranfected HEK293 cell line containing kidney cancer G250 gene and gene adjuvant hGM-CSF gene,provided a tool for further exploring the immune response mechanism of renal cell cancer （RCC） vaccine.
出处
《岭南现代临床外科》
2011年第4期270-273,共4页
Lingnan Modern Clinics in Surgery
基金
广东省十一五医学重点专科基金[2008(粤卫)50号]
