稳定表达EGFRvⅢex的NIH3T3细胞株的建立及其免疫原性分析

目的:建立表皮生长因子突变体Ⅲ胞外区(EGFRvⅡ-Iex)的NIH3T3稳定细胞系并分析其免疫原性。方法:将编码EGFRvⅢex基因的表达质粒pLNCX2-EGFRvⅢex转染NIH3T3细胞后,用G418筛选阳性克隆,得到多个细胞克隆。采用免疫组化和Western blot法鉴定这些细胞克隆。选取高表达EG-FRvⅢex的细胞株(命名为3T3-vⅢex)免疫BALB/c小鼠,制备抗血清。用ELISA、Western blot和免疫荧光分别检测抗血清的效价和特异性。结果:成功地构建了真核表达载体pLNCX2-EGFRvⅢex并获得了稳定高表达EGFRvⅢex的NIH3T3细胞系3T3-vⅢex。用3T3-vⅢex免疫小鼠所获得的抗血清效价为10-5。Western blot和免疫荧光鉴定证明抗血清可以与EGFR-vⅢex特异结合。结论:人EGFRvⅢex可在NIH3T3细胞内获得稳定表达,以其免疫小鼠可以获得高效价、高特异性的抗血清。

Establishment of a NIH3T3 cell line stably expressing EGFRvⅢ extracellular domain and evaluation of its immunogenicity

AIM: To establish a cell line stably expressing EGFRvIIIex (epidermal growth factor receptor variant III extracellular domain) and evaluate its immunogenicity. METHODS: NIH3T3 cells stably expressing EGFRvIIIex was obtained by screening NIH3T3 cells transfected with pLNCX2-EGFRvIIIex, a plasmid encoding EGFRvIIIex. The expression level of EGFRvIIIex was examined by immunohistochemical staining and Western blot. The cell clone with the highest expression of EGFRvIIIex named as 3T3-vIIIex was used to immunize BALB/c mice. The titer and specificity of murine antiserum was evaluated by ELISA, Western blot and immunofluorescent staining. RESULTS: A NIN3T3 stable cell line with high EGFRvIIIex expression was obtained. The titer of the antiserum against human EGFRvIIIex was about 10(-5). Western blot and immunofluorescent staining analysis revealed the high specificity of the antiserum. CONCLUSION: High titer of antiserum against EGFRvIIIex can be obtained by NIH3T3 cell lines stably expressing EGFRvIIIex.