PLA2R真核表达细胞株的构建及其在膜性肾病诊断中的应用  

Establishment of A Stable Cell Line CHO-PLA2R with High Expression of PLA2R and Its Application in the Diagnosis of Membranous Nephropathy

作　　者：王超男 李伟皓[2] 沈宝艳 李江雪 张佳 刘晓梅 张菲菲 张智萍 冯晓燕 张贺秋 WANG Chao-nan;LI Wei-hao;SHEN Bao-yan;LI Jiang-xue;ZHANG Jia;LIU Xiao-mei;ZHANG Fei-fei;ZHANG Zhi-ping;FENG Xiao-yan;ZHANG He-qiu(Medical Institute of Oriental Ocean(Beijing)Ltd.,Beijing 100071,China;Special Survey Division,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,China;Department of Nephropathy Immunology,the First Hospital of Mengcheng,Anhui Bozhou 233500,China)

机构地区：[1]东方海洋(北京)医学研究院,北京100071 [2]河北医科大学第二医院特检科,石家庄050000 [3]蒙城县第一人民医院肾病免疫风湿科,安徽亳州233500

出　　处：《现代检验医学杂志》2020年第4期1-5,13,共6页Journal of Modern Laboratory Medicine

基　　金：河北省卫生计生委,河北省2015年度医学科学研究重点课题计划(20150673)。

摘　　要：目的构建表达磷脂酶A2受体(phospholipase A2 receptor,PLA2R)的稳定细胞株并对其在膜性肾病(membranous nephropathy,MN)诊断中的应用进行初步评价。方法人工合成pIRES-PLA2R真核表达质粒并稳定转染CHO细胞,筛选后分别进行RT-PCR,Western免疫印迹和细胞免疫荧光方法(immunofluorescence assay,IFA法)鉴定。以构建的稳定细胞株建立PLA2R自身抗体IFA法并对2018~2019年收集的97例肾病患者血清样本进行初步检测。结果成功构建表达PLA2R蛋白的稳定细胞株CHO-PLA2R并以该细胞株为基础建立PLA2R自身抗体IFA法。与ELISA试剂盒检测结果相比,阴性样本符合率为100%(55/55);阳性样本符合率为76.2%(32/42);总体符合率为89.7%(87/97)。Kappa检验具有一定的一致性(P<0.05)。结论成功构建表达PLA2R蛋白的稳定细胞株,并应用该细胞株建立了检测PLA2R自身抗体的IFA检测方法。Objective To establish a stable cell line CHO-PLA2R with high expression of PLA2R and evaluated its application in the diagnosis of membranous nephropathy.Methods The eukaryotic expression plasmid pIRES-PLA2R was artificially constructed and stably transfected into CHO cells.The cell with expression of PLA2R was obtained after G-418 screening and identified by RT-PCR,western blotting and immunofluorescence respectively.Establishment of immunofluorenscence assay for PLA2R autoantibody and detection of the serum of 97 patients with nephropathy was performed.Results It was confirmed that CHO-PLA2R cell line was successfully constructed.An immunofluorescence assay for PLA2R autoantibody was successfully established utilizing the CHO-PLA2R cell.The preliminary evaluation showed that,compared with the results of ELISA method,the coincidence rate of negative samples was 100%(55/55),and positive samples was 76.2%(32/42),and the overall coincidence rate was 89.7%(87/97).There was no significant difference in Kappa test(P<0.05).Conclusion A stable cell line with high expression of PLA2R was successfully constructed,and a immunofluorescence assay for PLA2R autoantibody was established.

关 键 词：膜性肾病 磷脂酶A2受体 细胞株 质粒 免疫荧光方法 

分 类 号：R692[医药卫生—泌尿科学] R392.12[医药卫生—外科学]