HIV-1新发感染捕获酶联免疫试验重组抗原的研究

Development of a recombinant antigen for capture enzyme immunoassay to detect recent HIV-1 infection

目的构建、表达1型艾滋病病毒(HIV-1)重组蛋白,并尝试用于HIV-1新发感染捕获酶联免疫试验。方法设计、合成1种HIV-1重组蛋白(编号HIV-Ag-R03)的核酸序列,将它与pET30a-trx载体骨架连接、构建重组质粒。将重组质粒转化BL21(DE3)细胞,经异丙基硫代半乳糖苷诱导表达蛋白质。用重组蛋白包被酶标板,检测48份血浆样品(其中HIV-1抗体阳性、HIV阴性各24份),分析蛋白质的抗原性。将HIV-Ag-R03标记辣根过氧化物酶(HRP)后,取代BED捕获酶联免疫试验(BED CEIA)试剂盒中的BED肽及后续组分,比较2种方法检测18份HIV-1阳性样品的结果相关性。结果成功构建了HIV-Ag-R03的重组质粒,核酸序列确认无误。获得了包涵体表达的蛋白质,经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳分析大小与预测值一致。使用包被HIV-Ag-R03的间接酶联免疫试验检测48份1∶101稀释血浆样品,结果HIV-1抗体阳性样品的吸光度(OD)值(1.513±0.991)显著高于HIV阴性样品的OD值(0.022±0.008),差异有统计学意义(t=7.336,P<0.01)。用HIV-Ag-R03-HRP取代BED CEIA试剂盒中的BED肽及后续组分,调试HIV-Ag-R03-HRP的最佳稀释度为1∶2 500,18份HIV-1阳性样品的检测结果ODn值与BED CEIA的ODn值相关性好(r=0.939,P<0.01)。结论获得了1种具有抗原性的重组蛋白并成功标记HRP,建立了检测HIV-1新发感染的新模式。

Objective To construct and express an HIV-1 recombinant protein and attempt to use it in HIV-1 incidence capture enzyme immunoassay(CEIA).Methods The nucleic acid sequence for HIV-1 recombinant protein(named as HIV-Ag-R03)was designed and synthesized,and then ligated into the pET30 a-trx vector backbone to construct a recombinant plasmid.The recombinant plasmid was transformed into BL21(DE3)cells,and the protein was induced by isopropylthiogalactoside.Plasma specimens from 24 HIV-1 antibody positive and 24 HIV negative individuals were used to analyze the antigenicity of the recombinant protein.HIV-Ag-R03 labeled with horseradish peroxidase(HRP)was applied to replace the biotinylated BED peptide and subsequent components from the BED HIV-1 Capture Enzyme Immunoassay(BED CEIA)kit(Sedia Biosciences Corporation,USA).The results detecting recent infections in 18 HIV-1 positive specimens were compared with those by using the BED CEIA kit.Results The recombinant plasmid for HIV-Ag-R03 was successfully constructed,and the nucleic acid sequence was confirmed.Inclusion body of the recombinant protein was obtained,and the size of Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis analysis was consistent with the predicted value.An indirect enzyme immunoassay coated with HIV-Ag-R03 was used to detect 48 samples of 1∶101 diluted plasma specimens,and the absorbance(OD)values for HIV-1 antibody positive specimens(1.513±0.991)were significantly higher than those for HIV negative specimens(0.022±0.008),with the difference statistically significant(t=7.336,P<0.01).To replace the biotinylated BED peptide and subsequent components from the BED CEIA kit,the optimal dilution of HIV-Ag-R03-HRP was 1:2500.The ODn values for 18 HIV-1 positive specimens obtained with this new assay were correlated well with those by using BED CEIA kit(r=0.939,P<0.01).Conclusion An antigenic recombinant protein is obtained and successfully labeled with HRP for detecting recent HIV-1 infection with a novel design.