基于阻断膜金属蛋白酶TRABD2A的HIV储存库检测方法的建立  

Establishment of a membrane metalloproteinase TRABD2A blocking method for HIV reservoir detection

作　　者：欧阳嘉悦 王若琳 张晓维 梁国新[1] Ouyang Jiayue;Wang Ruolin;Zhang Xiaowei;Liang Guoxin(Department of Laboratory Medicine,The First Hospital of China Medical University,National Clinical Research Center for Laboratory Medicine,Shenyang 110001,China)

机构地区：[1]中国医科大学附属第一医院检验科、国家医学检验临床医学研究中心,沈阳110001

出　　处：《中华检验医学杂志》2023年第2期189-196,共8页Chinese Journal of Laboratory Medicine

基　　金：“十四五”国家重点研发计划(2021YFC2301900)。

摘　　要：目的探究人源化膜金属蛋白酶——含有TRAB结构域的蛋白2A(TRABD2A)单克隆抗体对接受联合抗逆转录病毒疗法(cART)的人免疫缺陷病毒(HIV-1)感染者储存库细胞的作用效果,建立基于TRABD2A阻断抗体的全新HIV-1储存库检测方法。方法2021年5—12月于中国医科大学附属第一医院收集研究对象51例。其中健康者2名,接受cART的HIV-1感染者(cART组)41例,未接受cART的HIV-1感染者(no-cART组)8例。采用噬菌体展示库技术构建人源化TRABD2A单克隆抗体,分别处理HIV-1感染者、未接受cART的HIV-1感染者及健康对照者的外周血单个核细胞(PBMC)和CD4+T淋巴细胞,利用荧光素酶报告系统、单分子免疫阵列检测技术等方法检测细胞培养上清中病毒含量,同时使用流式细胞术、荧光实时定量聚合酶链式反应检测处理后细胞的活化和病毒基因表达情况,比较不同处理组之间病毒释放量和表面活化标志CD25、CD69、HLA-DR(人类白细胞DR抗原)表达量的差异。结果接受cART的HIV-1感染者的PBMC经人源化TRABD2A单克隆抗体处理后检测HIV-1产量,未处理组为0(0,440),使用负抗体所释放的病毒量为0(0,390),二者差异无统计学意义(P>0.05),而使用正抗体所释放的病毒量为1259(0,4269)和3142(1292,5060),与使用负抗体所释放的病毒量相比,差异均有统计学意义(P<0.05)。利用健康对照者PBMC对感染者PBMC进行倍比稀释,经正抗体处理后病毒释放量等比例下降[未稀释、稀释5、25、125、625倍对应的HIV-1产量分别为4670(3339,7697)、1860(1509,4615)、1550(1150,2680)、602(255,1441)、2(0,37)]。且TRABD2A单抗处理后的细胞静息状态与未处理组差异无统计学意义(未处理组与正抗体-1处理组的CD25阳性细胞百分率分别为3.89±1.31、4.60±1.74,CD69阳性细胞百分率分别为2.50±1.27、2.18±0.51,HLA-DR阳性细胞百分率分别为7.66±3.78、8.79±3.42;P均>0.05),未处理与正抗体-1的病毒群抗原基因(Gag)表Objective To investigate the therapeutic effect of humanized TRAB domain-containing protein 2A(TRABD2A)monoclonal blocking antibody to HIV-1 reservoir cells,and to explore novel methods for measuring the sizes/capacities of HIV-1 infected reservoirs in HIV-1 infected individuals on receiving combined antiretroviral therapy(cART).Methods A total number of 51 subjects were collected from the First Affiliated Hospital of China Medical University from May 2021 to December 2021.Among them,there were 2 healthy persons,41 HIV-1 infected persons receiving cART(cART group)and 8 HIV-1 infected persons not receiving cART(no cART group).Humanized TRABD2A monoclonal antibody was constructed based on the phage display technology,the PBMCs and CD4+T cells separated from the peripheral blood mononuclear cells(PBMCs)and CD4+T cells of HIV-1 infected patients treated with receiving cART,or the HIV-1 infected patients without cART treatment and healthy controls were treated with TRABD2A monoclonal antibodies.The luciferase reporter system,single molecule immune array detection technology and other methods were used to detect the virus content in the supernatant of cell culture.At the same time,flow cytometry and fluorescence real-time quantitative polymerase chain reaction were used to detect the activation of the treated cells and the expression of virus genes.The statistical differences between different treatment the amount of virus release and the level of surface activation markers CD25,CD69,human leukocyte antigen DR(HLA-DR)of different groups in the amount of virus release and the expression of surface activation markers CD25,CD69,HLA-DR were compared.Results The PBMCs of HIV-1 infected persons receiving cART were tested for HIV-1 production after being treated with humanized TRABD2A monoclonal antibody.The amount of virus released by the untreated group was 0(0,440),and the amount of virus released by the use of negative antibody was 0(0,390).There was no significant difference between the two(P>0.05).The amount of virus re

关 键 词：人免疫缺陷病毒 潜伏 抗体 

分 类 号：R446[医药卫生—诊断学] R512.91[医药卫生—临床医学]