间日疟原虫醛缩酶的抗原和单克隆抗体制备及其在疟疾快速诊断中的应用
发布时间：2018-08-31 07:58
【摘要】：疟疾是全球死亡的一个主要病之一。大部分针对疟疾的快速诊断试剂不能区分来自于不同疟原虫的感染。开发灵敏度高且能区分两种最常见疟原虫感染的快速诊断试剂对于疾病的控制和治疗有重要的意义。本文以提取的间日疟原虫DNA为模板,通过PCR扩增了醛缩酶基因(PvALDO)并成功构建pET30a-Aldo原核表达质粒,成功在大肠杆菌BL21(DE3)中表达并使用亲和层析法纯化得到了纯度高于95%的重组间日疟原虫醛缩酶蛋白。用重组蛋白对小鼠和兔子进行免疫用于生产抗体。将生产得到的兔多克隆抗体使用在一种新型抗体捕获ELISA法进行杂交瘤细胞的筛选,一共筛选得到九个稳定的单克隆抗体(1C3,1G3,1H3,3F1,9A1,9B5,9G6,11G7,12F10),将得到的单克隆抗体用作包被抗体或者标记抗体在胶体金免疫层析平台进行评价。 把实验结果最好的抗体对(1C3-12F10)用于开发商业化的间日疟原虫检测快速诊断试剂并对其检测效果进行评估。对来自云南和广州的临床血液样本(n=503)的评估结果表明我们的快速诊断试剂针对间日疟原虫的特异性为100%(95%Confidence Interval (CI):99.12to100.00%),与显微镜镜检法相比灵敏度为98.75%(95%CI:93.20to99.79%)且其中仅出现一例假阴性。同时对恶性疟原虫样本(n=33)和健康人血液样本(n=390)的检测没有发应。我们的快速诊断试剂的阳性预测值(PPV)以及阴性预测值(NPV)分别为100.00%和99.76%,检测的置信区间为95%,与显微镜镜检法有很好的相关性(kappa值,K=0.992530).针对重组间日疟原虫醛缩酶蛋白,胶体金检测试纸条的灵敏度能达到6.25ng/ml。本文阐明了以醛缩酶蛋白作为检测指标的快速诊断试剂区分不同疟原虫引起的疟疾感染是可行的。开发得到的快速诊断试剂能有效区分间日疟原虫感染和恶性疟原虫感染,同时本文采用的新的抗体筛选方法可以在其他抗原高特异性单克隆抗体的筛选过程中得到运用。
[Abstract]:Malaria is one of the leading causes of death worldwide. Most rapid diagnostic kits for malaria do not distinguish between infections from different malaria parasites. It is of great significance to develop a rapid diagnostic reagent with high sensitivity and ability to distinguish the two most common Plasmodium infections for the control and treatment of the disease. Based on the extracted DNA of Plasmodium vivax, the aldolase gene (PvALDO) was amplified by PCR and the pET30a-Aldo prokaryotic expression plasmid was constructed successfully. The recombinant plasmodium vivax aldolase protein was successfully expressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. Mice and rabbits were immunized with recombinant proteins to produce antibodies. Rabbit polyclonal antibodies were used in a novel antibody capture ELISA method to screen hybridoma cells. A total of nine stable monoclonal antibodies (1C3G3G3H3H3F1F1F1F1A1A1A1F10) were obtained. The monoclonal antibodies were used as coated or labeled antibodies for evaluation on a colloidal gold immunochromatographic platform. The best antibody pair (1C3-12F10) was used to develop a commercial rapid diagnostic reagent for Plasmodium vivax and to evaluate its effectiveness. The evaluation results of clinical blood samples from Yunnan and Guangzhou showed that the specificity of our rapid diagnostic reagent for Plasmodium vivax was 100% (95%Confidence Interval (CI): 99.12 to 100.00%), and the sensitivity was 98.75% (95CIW 93.20 to 99.79%) compared with microscopic examination. At the same time, the detection of Plasmodium falciparum (nm33) and healthy blood samples (nm390) was not detected. The positive predictive value (PPV) and negative predictive value (NPV) of our rapid diagnostic reagent were 100.00% and 99.76%, respectively, and the confidence interval was 95, which had a good correlation with microscopic examination (kappa value: 0.992530). For recombinant Plasmodium vivax aldolase protein, the sensitivity of colloidal gold test strip was 6.25 ng / ml. It is feasible to distinguish malaria infection caused by different malaria parasites by using aldolase protein as a rapid diagnostic reagent. The developed rapid diagnostic reagent can effectively distinguish the infection of Plasmodium vivax from that of Plasmodium falciparum. At the same time, the new antibody screening method used in this paper can be used in the screening process of other highly specific monoclonal antibodies against Plasmodium vivax and Plasmodium falciparum.
【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】：R531.3;Q78
本文编号：2214374
[Abstract]:Malaria is one of the leading causes of death worldwide. Most rapid diagnostic kits for malaria do not distinguish between infections from different malaria parasites. It is of great significance to develop a rapid diagnostic reagent with high sensitivity and ability to distinguish the two most common Plasmodium infections for the control and treatment of the disease. Based on the extracted DNA of Plasmodium vivax, the aldolase gene (PvALDO) was amplified by PCR and the pET30a-Aldo prokaryotic expression plasmid was constructed successfully. The recombinant plasmodium vivax aldolase protein was successfully expressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. Mice and rabbits were immunized with recombinant proteins to produce antibodies. Rabbit polyclonal antibodies were used in a novel antibody capture ELISA method to screen hybridoma cells. A total of nine stable monoclonal antibodies (1C3G3G3H3H3F1F1F1F1A1A1A1F10) were obtained. The monoclonal antibodies were used as coated or labeled antibodies for evaluation on a colloidal gold immunochromatographic platform. The best antibody pair (1C3-12F10) was used to develop a commercial rapid diagnostic reagent for Plasmodium vivax and to evaluate its effectiveness. The evaluation results of clinical blood samples from Yunnan and Guangzhou showed that the specificity of our rapid diagnostic reagent for Plasmodium vivax was 100% (95%Confidence Interval (CI): 99.12 to 100.00%), and the sensitivity was 98.75% (95CIW 93.20 to 99.79%) compared with microscopic examination. At the same time, the detection of Plasmodium falciparum (nm33) and healthy blood samples (nm390) was not detected. The positive predictive value (PPV) and negative predictive value (NPV) of our rapid diagnostic reagent were 100.00% and 99.76%, respectively, and the confidence interval was 95, which had a good correlation with microscopic examination (kappa value: 0.992530). For recombinant Plasmodium vivax aldolase protein, the sensitivity of colloidal gold test strip was 6.25 ng / ml. It is feasible to distinguish malaria infection caused by different malaria parasites by using aldolase protein as a rapid diagnostic reagent. The developed rapid diagnostic reagent can effectively distinguish the infection of Plasmodium vivax from that of Plasmodium falciparum. At the same time, the new antibody screening method used in this paper can be used in the screening process of other highly specific monoclonal antibodies against Plasmodium vivax and Plasmodium falciparum.
【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】：R531.3;Q78
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