布鲁氏菌病新的免疫学诊断靶点的研究
发布时间：2018-11-01 14:40
【摘要】：【目的】 布鲁氏菌病（Brucellosis，简称布病）是一种全世界广泛分布的人兽共患慢性细菌传染病，由布鲁氏菌（Brucella）感染机体引起，人间布鲁氏菌病在我国被列为法定乙类传染病。传统的布鲁氏菌病实验室检测主要依赖于病原体的分离，该方法耗时耗力，且需专业的实验室，并不实用；随着技术的发展，发现酶联免疫吸附实验（ELISA）方法与补体结合实验（CFT）、传统的试管凝集实验（SAT）相比，有更高的敏感度和特异度。本研究试图选用布鲁氏菌中具有代表性的重组蛋白作为抗原进行酶联免疫吸附实验（ELISA），从而建立以重组蛋白做为抗原的ELISA方法，为研制快速诊断布鲁氏菌病的ELISA试剂盒奠定基础。 【方法】 通过参考大量关于布鲁氏菌相关的国内外文献，综合云南省地方病防治所中心实验室对布鲁氏菌诊断靶点的研究情况，选取外膜蛋白omp10，核蛋白L7/12为诊断靶点进行初步研究。 1.布鲁氏菌omp10、L7/L12的克隆表达、纯化 根据已发表的布鲁氏菌外膜蛋白omp10、核蛋白L7/L12基因设计两对引物进行扩增，用BamHⅠ和NotⅠ或EcoRⅠ和NotⅠ将目的基因片段双酶切纯化与同样双酶切纯化的载体PGEX-4T-1或PET32a（㧏）连接，之后将重组表达载体转化到大肠杆菌BL21中。重组蛋白经菌落PCR鉴定、双酶切鉴定、Western-blot鉴定。应用AKTA purifier色谱系统对重组蛋白进行纯化分析。 2.ELISA检测方法的建立 用纯化后omp10和L7/L12两种重组蛋白做抗原对人、牛血清进行检测，以验证两种蛋白用ELISA方法检测人、牛布鲁氏菌病的应用价值。通过对纯化蛋白浓度的测定，，确定不同包被浓度抗原的包被量后，进行方阵滴定法确定ELISA最佳包被浓度。 【结果】 本实验成功构建了含有布鲁氏菌外膜蛋白omp10和核蛋白L7/L12序列的原核表达载体PET32a/Omp10、PGEX-4T-1/Omp10和PGEX-4T-1/L7/L12，并经过PCR、双酶切鉴定，均证明确实插入了表达载体。利用IPTG诱导，PGEX-4T-1/Omp10和PGEX-4T-1/L7/L12在其宿主菌大肠杆菌BL21中均有蛋白表达，经纯化后以重组蛋白PGEX-4T-1/L7/L12和PGEX-4T-1/omp10做为抗原进行的ELISA实验结果表明这两种重组蛋白对人布鲁氏菌病的检测没有实际的应用价值，但对检测动物布鲁氏菌病有一定潜力。
[Abstract]:[objective] brucellosis (Brucellosis,) is a chronic bacterial infection caused by brucella (Brucella) infection, which is widely distributed in the world. Human brucellosis is classified as a class B infectious disease in China. The traditional laboratory detection of brucellosis mainly depends on the isolation of pathogens. This method is time-consuming and labor-intensive and requires a professional laboratory, which is not practical. With the development of the technology, it was found that the Elisa (ELISA) method had higher sensitivity and specificity than the conventional (CFT), agglutination test (SAT). In this study, the representative recombinant protein of Brucella was selected as antigen to carry out the enzyme linked immunosorbent assay (ELISA),) to establish a ELISA method with recombinant protein as antigen. It lays a foundation for the development of ELISA kit for rapid diagnosis of brucellosis. [methods] by referring to a large number of domestic and foreign literatures on brucellosis, and synthesizing the research on the diagnostic targets of brucella in the central laboratory of Yunnan Provincial Institute of endemic Disease Prevention and Control, the outer membrane protein omp10, was selected. Nuclear protein L 7 / 12 was used as a diagnostic target. 1. Cloning and expression of Brucella omp10,L7/L12, purification and amplification of two pairs of primers based on the published L7/L12 gene of omp10, nucleoprotein of Brucella spp. The vector PGEX-4T-1 or PET32a (?) was digested by BamH 鈪
本文编号：2304227
[Abstract]:[objective] brucellosis (Brucellosis,) is a chronic bacterial infection caused by brucella (Brucella) infection, which is widely distributed in the world. Human brucellosis is classified as a class B infectious disease in China. The traditional laboratory detection of brucellosis mainly depends on the isolation of pathogens. This method is time-consuming and labor-intensive and requires a professional laboratory, which is not practical. With the development of the technology, it was found that the Elisa (ELISA) method had higher sensitivity and specificity than the conventional (CFT), agglutination test (SAT). In this study, the representative recombinant protein of Brucella was selected as antigen to carry out the enzyme linked immunosorbent assay (ELISA),) to establish a ELISA method with recombinant protein as antigen. It lays a foundation for the development of ELISA kit for rapid diagnosis of brucellosis. [methods] by referring to a large number of domestic and foreign literatures on brucellosis, and synthesizing the research on the diagnostic targets of brucella in the central laboratory of Yunnan Provincial Institute of endemic Disease Prevention and Control, the outer membrane protein omp10, was selected. Nuclear protein L 7 / 12 was used as a diagnostic target. 1. Cloning and expression of Brucella omp10,L7/L12, purification and amplification of two pairs of primers based on the published L7/L12 gene of omp10, nucleoprotein of Brucella spp. The vector PGEX-4T-1 or PET32a (?) was digested by BamH 鈪
本文编号：2304227
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