人巨细胞病毒PP65抗原单克隆抗体的制备及鉴定  

Preparation and characterization of monoclonal antibodies against human cytomegalovirus PP65 antigen

作　　者：张淑婷 田海霞 刘青青[1] 康行 樊卫平[1] ZHANG Shuting;TIAN Haixia;LIU Qingqing;KANG Xing;FAN Weiping(Department of Microbiology and Immunology,Shanxi Medical University,Taiyuan,Shanxi Province,030001,China)

机构地区：[1]山西医科大学微生物学与免疫学教研室,太原030001

出　　处：《第三军医大学学报》2020年第12期1182-1187,共6页Journal of Third Military Medical University

基　　金：山西省专利推广实施资助专项(20171009);山西省科技开发基金(033068)。

摘　　要：目的制备人巨细胞病毒(human cytomegalic virus,HCMV)PP65抗原的单克隆抗体,并通过免疫组化鉴定单克隆抗体与HCMV PP65抗原的反应性。方法诱导PQE30-PP65重组质粒表达PP65蛋白并Ni柱亲和层析纯化。PP65抗原免疫BALB/c小鼠,将血清抗体阳性小鼠脾细胞与骨髓瘤细胞(SP2/0)融合,经HTS筛选,扩大培养获取单克隆抗体。经Protein A亲和层析纯化,并通过间接ELISA鉴定其特异性、效价及亚型。将制备的单克隆抗体应用于临床样本的玻片免疫组化检测。结果获得8株高特异性抗HCMV PP65抗原的单克隆抗体,其亚型均为IgG1型,轻链均为κ链,效价均在1∶2048000以上。其中6株单克隆抗体能够通过玻片免疫组化法准确鉴定HCMV激活性感染。结论成功制备了HCMV PP65单克隆抗体。单抗与白细胞中HCMV PP65抗原反应良好。Objective To prepare monoclonal antibodies(mAbs)against human cytomegalovirus(HCMV)PP65 antigen and characterize their reactivity to HCMV PP65 antigen by immunohistochemistry.Methods The recombinant plasmid PQE30-PP65 was transformed into competent E.coli M15 cells to obtain the recombinant PP65 antigen,which was purified with Ni column affinity chromatography.BALB/c mice were immunized with the purified antigen,and the spleen cells of the mice were isolated and fused with myeloma cells(SP2/0).The mAbs were obtained by HTS screening and purified by protein A affinity chromatography,and their specificity,titer and subtype were identified by indirect ELISA.The performance of the mAbs in immunohistochemical detection of HCMV PP65 antigen was tested on slides of clinical samples.Results Eight highly specific mAbs against HCMV PP65 antigen were obtained.All these mAbs were identified to be IgG1 subtype containingκlight chains,with titers all above 1∶2048000.Six of the mAbs were shown to accurately identify an active infection of HCMV by slide immunohistochemistry.Conclusion We successfully prepared HCMV PP65 mAbs,and 6 of these mAbs have good reactivity with HCMV PP65 antigen in leukocytes.

关 键 词：人巨细胞病毒 PP65抗原 单克隆抗体 免疫组化 

分 类 号：R373.9[医药卫生—病原生物学] R392-33[医药卫生—基础医学]