基于FRET的p24抗原表位均相免疫探针构建及其在HIV抗体快速检测中的应用

Construction of a p24 epitope homogeneous immunoprobe based on FRET and its application in rapid clinical detection of anti-HIV antibodies

目的基于基因编码的Förster共振能量转移(FRET)技术构建HIV p24抗原表位荧光蛋白探针并进行临床验证,为开发快速检测HIV p24抗体的均相免疫检测试剂奠定基础。方法以荧光蛋白ECFP、EYFP作为FRET的供体和受体,将p24抗原表位对应的核酸序列插入ECFP-Linker-EYFP探针骨架,采用定点突变构建p24荧光蛋白二聚化界面,通过pET28a载体构建其重组质粒,进而转化至E.coli BL21感受态细胞中诱导表达p24抗原表位荧光蛋白探针,并采用Ni柱及分子筛对探针进行纯化。筛选探针的最佳浓度,检测探针与p24多克隆抗体的结合能力及光谱学变化。选择60例HIV阳性患者血清和58例正常人血清,通过探针进行检测,通过特异性、敏感度、阳性预测值、阴性预测值和诊断效率等评价探针对HIV p24抗体的检测效能。结果高通量测序结果显示p24抗原表位荧光蛋白探针构建成功,其理论分子量约为67kDa,最佳浓度为0.20 mg/mL。探针在结合p24多克隆抗体后在530/477 nm处荧光强度比值降低(2.13vs.1.47)。HIV阳性患者血清530/477 nm比值(1.42±0.21)明显小于正常人(1.76±0.11)(P<0.001),两组比值进行ROC曲线分析,得到曲线下面积为0.97,标准误0.02,P<0.001。分析结果表明,p24抗原表位荧光蛋白探针临床检测HIV p24抗体的敏感度为88.33%,特异性为100.00%,阳性预测值88.33%,阴性预测值100.00%,诊断效率为94.07%。结论成功构建基于FRET的p24抗原表位均相免疫检测探针,用于HIV/AIDS患者HIV p24抗体的检测初步显示出了较好的敏感性与特异性,明显缩短了检测时间,为进一步提高HIV p24抗体实验室检测的灵敏度和时效性提供了思路和依据。

Objective To construct and clinically validate a fluorescent protein probe for HIV p24 antigenic epitope detection based on genetically encoded fluorescence resonance energy transfer(FRET)technology,laying the foundation for the development of a homogeneous immunoassay reagent for the rapid detection of anti-HIV P24 antibodies.Methods The fluorescent proteins ECFP and EYFP were used as FRET donors and acceptors,respectively,and nucleic acid sequences corresponding to p24 antigenic epitopes were inserted into an ECFP-Linker-EYFP probe backbone.A p24 fluorescent protein dimerization interface was constructed using targeted mutagenesis,and a recombinant plasmid was constructed using the pET28a vector,which was transformed into E.coli BL21 recipient cells to induce the expression of p24 antigenic epitope fluorescent protein probes,and purified using Ni columns and molecular sieves.The optimal concentration of the probe was screened,and the binding ability and spectroscopic changes of the probe to anti-p24 polyclonal antibodies were detected.Sixty sera from HIV-positive patients and 58 sera from normal subjects were selected for detection by the probe,and the efficacy of the probe for the detection of anti-HIV p24 antibody was evaluated by specificity,sensitivity,positive predictive value,negative predictive value,and diagnostic efficiency.Results Highthroughput sequencing results demonstrated that the p24 antigen epitope fluorescent protein probe was successfully constructed,its theoretical molecular weight was approximately 67 kDa,and the optimal concentration was 0.20 mg/mL.The fluorescence intensity ratio at 530/477 nm was reduced after binding the p24 polyclonal antibody to the probe(2.13 vs.1.47).The 530/477 nm ratio(1.42±0.21)of HIV-positive patient sera was significantly lower than that of normal patients(1.76±0.11),P<0.001.ROC curve analysis of the ratio between the two groups yielded an area under the curve of 0.97 with a standard error of 0.02,P<0.001.The sensitivity of the p24 antigenic epitope fluorescent protein probe for the clinical detection of the HIV p24 antibody was 88.33%,and the specificity was 100.00%.The positive predictive value was 88.33%,the negative predictive value was 100.00%,and the diagnostic efficiency was 94.07%.Conclusions The successful construction of a FRET-based p24 antigen epitope homogeneous immunoassay probe for the detection of anti-HIV p24 antibody in HIV/AIDS patients exhibited good sensitivity and specificity,significantly shortened the detection time,and provided insights for further improvements in sensitivity and timeliness of anti-HIV p24 antibody laboratory testing.