hFcεRIα/RBL-2H3细胞株的构建与评价

Construction and identification of recombinant RBL-2H3 cells transfected with hFcεRIα

目的构建稳定表达人FcεRIα(h FcεRIα)亚基的RBL-2H3细胞株。方法利用RT-PCR技术克隆h FcεRIα基因,构建真核表达载体p CI-neo-h FcεRIα。脂质体介导法转染RBL-2H3细胞,G418筛选获得稳定转染细胞株。利用RT-PCR、Western blot及免疫荧光法鉴定转染结果。结果通过对脂质体介导转染体系进行优化,转染效率可高达75.38%。经Western blot、免疫荧光及RT-PCR鉴定,h FcεRIα基因在RBL-2H3细胞内成功表达。结论成功构建h FcεRIα/RBL-2H3细胞模型,为深入研究Ig E与FcεRI作用机制及相关药物开发提供了实验基础。

Aim To construct the stable hFcεRIα/RBL-2H3 cell line expressing human FcεRIα（hFcaRIct）. Methods The human FcεRIα gene was obtained by RT-PCR and cloned into the eukaryotic expression vector pCI-neo. Then, the pCI-neo- hFcεRIα vector was transfected into RBL-2H3 cells by lipo- somes, and the transfected cells were screened through G418 fil- tration subsequently. Finally, RT-PCR, Western blot and immu- nofiuoreseence assay were used to determine the result of trans- fection. Results According to the optimized transfection param-eters, the transfection efficiency reached 75.38%. The results of Western blot, immunofluoreseence and RT-PCR showed that hFcεRIα could be expressed in RBL-2H3 cells successfully. Conclusion HFeεRIα/RBL-2H3 cells were successfully con- structed, which will be the experimental basis for further study on the mechanism of IgE/FcεRI and drugs for allergy diseases.