Establishment of MDCK cell lines which stably express visualable human neonatal Fc receptor
- 公共卫生－已发表论文 
[目的]建立稳定表达融合EGFP的人新生儿Fc受体（h FcRn）的MDCK细胞株。[方法]构建重组慢病毒质粒p EGFP-h FcRn,采用四质粒包装系统共转染HEK 293T细胞生产重组慢病毒,感染MDCK细胞后对EGFP阳性细胞进行流式单细胞分选;通过Western Blot及EGFP-β2m荧光共定位验证h FcRn的完整性,并用流式细胞仪检测h FcRn与人Ig G的结合活性。[结果]测序结果表明成功构建p EGFP-FcRn慢病毒表达载体;感染后EGFP阳性MDCK细胞比例约为26.5%,流式单细胞分选后得到纯阳性细胞;荧光共定位及Western Blot均检测到h FcRn的完整表达;流式分析表明细胞株上的h FcRn与Ig G存在p H依赖性结合。[结论]成功获得稳定表达具有生物活性的可视化h FcRn的MDCK细胞株。[ Objective] To establish MDCK cell line stably expressing EGFP- human neonatal Fc receptor（hFcRn） fusion protein. [ Methods ] The lentiviral expression vector for EGFP - hFcRn fusion protein was constructed. Generating by co - transfection of four -plasmids into HEK 293T cells ,the lentivirus particles were used to infect MDCK cell line. EGFP positive single cell was obtained by FACS, and then FcRn expression was identified by fluorescence co -location with EGFP - β2m and confirmed by Western Blot. Flow cytometry was used to detect binding activity of hFcRn and human IgG. [ Results ] DNA se- quencing demonstrated that the lentivirus vector pEGFP - FcRn was constructed successfully. The percentage of EGFP - posi- tive ceils was about 26.5% after infection. Expression of the complete protein was detected through fluorescence co - location and Western Blot, respectively. Flow cytometry analysis showed that the cell lines could pH - dependently capture human IgG. [ Conclusion] MDCK cell line stably expressing functional visualable hFcRn was established.