高表达FcεR1α和共表达NPY-EGFP的RBL细胞系构建及生物学功能检测  

Construction of RBL cell lines with high expression of FcεR1α and co-expression of NPY-EGFP and identification of its biological functions

作　　者：黄伦辉 张嘉懿[3] 李柳栩 张蓓 刘运德[1] 李会强[1] HUANG Lun-hui;ZHANG Jia-yi;LI Liu-xu;ZHANG Bei;LIU Yun-de;LI Hui-qiang(College of Medical Laboratory,Tianjin Medical University,Tianjin300203,China;State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences,Tianjin300020,China;Department of Medical Laboratory,Tianjin Children's Hospital,Tianjin300074,China)

机构地区：[1]天津医科大学医学检验学院,天津300203 [2]中国医学科学院血液病医院(中国医学科学院血液学研究所)实验血液学国家重点实验室,国家血液系统疾病临床医学研究中心,天津300020 [3]天津市儿童医院检验科,天津300074

出　　处：《现代免疫学》2021年第2期89-93,共5页Current Immunology

基　　金：国家自然科学基金(81772259)。

摘　　要：为构建一种高表达高亲和力IgE受体α链(high affinity IgE receptor alpha chain, FcεR1α)和共表达新型标志物神经肽Y(neuropeptide Y, NPY)-增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)的RBL-2H3细胞模型并鉴定其活性,设计FcεR1α-NPY-EGFP序列,构建表达载体pLV-Puro-FcεR1α-T2A-NPY-EGFP-TYK2,对其进行测序鉴定后无内毒素大量提取;经脂质体Lipo2000瞬时转染,于48 h后通过荧光定量PCR、Western blotting、FACS鉴定FcεR1α表达水平;通过传统β-氨基己糖苷酶介质释放试验检测受体的活性,通过NPY-EGFP释放试验检测新型指标的功能。经基因水平、蛋白质水平、膜蛋白表达及功能试验鉴定FcεR1α位于RBL-2H3细胞膜上并有结合人特异性IgE(specific IgE, sIgE)的活性,新型标志物NPY-EGFP可以受刺激分泌。该研究成功构建了一种高表达FcεR1α和共表达新型标志物NPY-EGFP的细胞模型用于检测人血清sIgE。To develop a RBL-2 H3 cell line with high expression of high affinity IgE receptor alpha chain(FcεR1α) and co-expression of neuropeptide Y(NPY)-enhanced green fluorescent protein(EGFP) and identify its activity, the expression vector pLV-Puro-FcεR1α-T2 A-NPY-EGFP-TYK2 was constructed by designing FcεR1α-NPY-EGFP gene. After sequencing, endo-free plasmid DNA was extracted in large quantity. After 48 hours transfection with Lipo2000, the expression of FcεR1α was identified by real-time fluorescence quantification PCR, Western blotting and FACS. Beta aminohexosaminase mediator release assay was used to appraise the receptor activity, and the NPY-EGFP release assay was used to verify the target function. Expression of FcεR1α on RBL-2 H3 cell membrane was identified at the levels of gene, protein and membrane protein expression. Functional studies demonstrated the binding of membrane FcεR1α to human specific IgE(sIgE) and the new marker NPY-EGFP could be secreted upon stimulation. Thus, a cell model with high expression of FcεR1α and co-expression of NPY-EGFP marker had been developed for the detection of sIgE in human serum.

关 键 词：细胞系 高亲和力IgE受体α链 神经肽Y-增强型绿色荧光蛋白 特异性IGE 

分 类 号：R446.61[医药卫生—诊断学]