CAJ | 학술논문

目的构建朗格汉斯细胞特异性C型凝集素受体Langerin体外细胞表达模型.方法 PCR方法获得Langerin分子的cDNA,克隆入真核绿色荧光蛋白(EGFP)表达载体pEGFP-C1(EGFP位于Langerin的N末端),转染人肾胚细胞癌HEK 293T细胞后,激光共聚焦显微镜检测EGFP-Langerin融合蛋白的细胞表达情况,流式细胞仪检测Langerin受体的表达,并进一步检测转染表达的Langerin受体对尘螨抗原(nDerp 2)的识别和内吞情况.结果构建的荧光融合蛋白重组表达质粒转染HEK 293T细胞后,经PCR及Western印迹检测证明Langerin转染及表达成功.重组表达质粒转染HEK293T细胞后,经流式细胞仪检测转染Langerin融合质粒的HEK293T细胞的Langerin受体表达率较转染前增多约43％,经激光共聚焦显微镜检测显示绿色荧光标记的Langerin成功表达,并可与红色荧光素标记的尘螨抗原(nDerp 2)结合,将nDer p 2内吞入细胞内.结论构建的EGFP-Langerin融合蛋白在HEK293T细胞内表达后具有细胞表面受体分子的特征性分布,具有结合、内吞过敏原功能.
목적 구건랑격한사세포특이성C형응집소수체Langerin체외세포표체모형.방법 PCR방법획득Langerin분자적cDNA,극륭입진핵록색형광단백(EGFP)표체재체pEGFP-C1(EGFP위우Langerin적N말단),전염인신배세포암HEK 293T세포후,격광공취초현미경검측EGFP-Langerin융합단백적세포표체정황,류식세포의검측Langerin수체적표체,병진일보검측전염표체적Langerin수체대진만항원(nDerp 2)적식별화내탄정황.결과 구건적형광융합단백중조표체질립전염HEK 293T세포후,경PCR급Western인적검측증명Langerin전염급표체성공.중조표체질립전염HEK293T세포후,경류식세포의검측전염Langerin융합질립적HEK293T세포적Langerin수체표체솔교전염전증다약43％,경격광공취초현미경검측현시록색형광표기적Langerin성공표체,병가여홍색형광소표기적진만항원(nDerp 2)결합,장nDer p 2내탄입세포내.결론 구건적EGFP-Langerin융합단백재HEK293T세포내표체후구유세포표면수체분자적특정성분포,구유결합、내탄과민원공능.
Objective To establish a cell model expressing the Langerhans cell-specific C type lectin receptor Langerin in vitro.Methods The cDNA of Langerin was obtained by PCR and cloned into a eukauotic green fluorescent protein (EGFP) expression vector pEGFP-C 1 with EGFP located in the N terminal region of the Langerin gene.Then,the recombinant plasmid was transfected into a human embryonic kidney carcinoma cell line HEK293T.Subsequently,laser confocal microscopy was performed to observe the expression of EGFP-Langerin fusion protein,and flow cytometry to measure the expression of Langerin.Laser confocal microscopy was also conducted to visualize the recognition and endocytosis of dust mite antigen (nDer p 2) by Langerin.Results PCR and Western blot confirmed the successful transfection of HEK293T cells with the recombinant plasmid as well as the expression of Langerin in the transfected cells.As flow cytometry revealed,the expression level of Langerin in transfected HEK293T cells was increased by 43％ compared with untransfected cells.Laser confocal microscopy showed that green fluorescein-labeled Langerin was successfully expressed,and could bind to and endocytose the red fluorescein-labeled antigen nDer p 2.Conclusions The fusion protein EGFP-Langerin expressed in HEK293T cells showed the distribution characteristic of cell surface receptors,and could bind to and endocytose allergens.