HIV-1核心蛋白p24单克隆抗体的制备及其在免疫检测中的应用

发布时间：2018-02-25 15:36

本文关键词： 人类免疫缺陷病毒单克隆抗体胶体金免疫层析 p24检测酶联免疫吸附测定　出处：《华南理工大学》2013年硕士论文　论文类型：学位论文

【摘要】：P24蛋白是1型人类免疫缺陷病毒(HIV-1)的衣壳蛋白,在病毒的包装和成熟过程中起着重要的作用。P24蛋白在HIV-1感染者感染早期和晚期的血清中含量相对较高,因此p24蛋白被认为是HIV-1早期检测、献血筛查以及监测疾病进程的重要指标。本文根据数据库中p24蛋白的氨基酸序列,设计并合成编码基因且成功构建pET28a-p24原核表达质粒,成功在大肠杆菌BL21(DE3)中表达并用亲和层析纯化得到了纯度高于95%的重组p24蛋白,间接酶联免疫吸附测定(ELISA)结果表明重组p24蛋白可被抗p24抗原的单克隆抗体识别。使用重组p24蛋白免疫Balb/c小鼠,将免疫后血清效价达到融合要求的小鼠的脾细胞与SP2/0骨髓瘤细胞融合制备杂交瘤细胞。以p24蛋白作为抗原进行间接ELISA法筛选,经多次有限稀释法亚克隆得到33株稳定分泌抗p24抗原的抗体的杂交瘤细胞株,制备腹水并用辛酸硫酸铵联合沉淀法或Protein G注亲和层析法纯化单克隆抗体。将得到的单克隆抗体进行ELISA平台的双抗体夹心配对,接着将得到的34种能在ELISA平台配对的抗体组合应用于胶体金免疫层析检测(GICA)平台的p24抗原检测,最终筛选出灵敏度和特异性相对最高的单克隆抗体组合3-1D5和4-8A10,该抗体组合在GICA平台对p24抗原的最低检测浓度为25pg/mL。利用3-1D5和4-8A10抗体组合在GICA平台检测HIV-1p24抗原国家参考品,对其中全部的10个阳性参考品均检出阳性并可以检测参考品中浓度低至5U/mL的WHO推荐p24蛋白标准品,表现出较高的灵敏度。再检测HIV抗原抗体均为阴性的血清样品(n=153),其中出3份假阳结果,显示出了良好的特异性。实验结果表明用制备得到的单克隆抗体组合开发GICA法p24抗原快速检测有很好的应用前景,为HIV第四代快速检测试剂的研发打下了坚实的基础。
[Abstract]:P24 protein is the capsid protein of human immunodeficiency virus type 1 (HIV-1). P24 protein plays an important role in the packaging and maturation of the virus. P24 protein is relatively high in the early and late stage of HIV-1 infection. Therefore, p24 protein is considered to be an important indicator of early detection of HIV-1, screening of blood donors and monitoring of disease progression. According to the amino acid sequence of p24 protein in the database, the encoding gene was designed and synthesized, and the prokaryotic expression plasmid of pET28a-p24 was successfully constructed. The recombinant p24 protein with purity of more than 95% was obtained by affinity chromatography. Indirect enzyme-linked immunosorbent assay (Elisa) showed that the recombinant p24 protein could be recognized by monoclonal antibodies against p24 antigen. Balb/c mice were immunized with recombinant p24 protein. Hybridoma cells were prepared by fusion of spleen cells of mice with SP2/0 myeloma cells whose titer of serum reached fusion requirement after immunization. P24 protein was used as antigen for indirect ELISA screening. 33 hybridoma cell lines secreting antibodies against p24 antigen were obtained by multiple limited dilution subcloning. Preparation of ascites and purification of monoclonal antibodies with ammonium octanoate combined with precipitation or Protein G affinity chromatography. The obtained monoclonal antibodies were paired with double antibodies of ELISA platform. Then, 34 kinds of antibody combinations which can be paired on ELISA platform were used to detect p24 antigen of GICA platform by colloidal gold immunochromatography. Finally, the relative highest sensitivity and specificity of monoclonal antibody combinations 3-1D5 and 4-8A10 were screened. The minimum detection concentration of p24 antigen on GICA platform was 25pg / mL. using the combination of 3-1D5 and 4-8A10 antibodies to detect HIV-1p24 antigen in GICA platform, All of the 10 positive reference samples were positive and could be used to detect p24 protein standard samples with a concentration as low as 5 U / mL in the reference. The serum samples with negative HIV antigen and antibody were all negative, and 3 of them had false positive results. The experimental results show that the rapid detection of p24 antigen by GICA method using the prepared monoclonal antibody combination has a good application prospect and lays a solid foundation for the development of rapid detection reagent for HIV 4th generation.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.91;R392

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