基于多表位融合蛋白的人布鲁氏菌病检测技术研究

发布时间：2018-05-10 04:56

本文选题：布鲁氏菌病 + 融合蛋白　；参考：《吉林大学》2016年博士论文

【摘要】：布鲁氏菌病是一种再发的人兽共患传染病,近年来发病率呈逐年上升的趋势,全球每年超过500 000新增病例,我国发病人数也在逐年增长,2015年全国新增病例60 000例。布鲁氏菌病不仅会造成感染动物流产,给畜牧业带来巨大经济损失,而且会给人类的健康造成巨大危害。人类感染布鲁氏菌病后,患者会出现发热、肝脾肿大、骨关节病及血液学改变等临床症状,严重时会造成孕妇流产、新生儿死亡或者出生体重降低。对布鲁氏菌病的诊断、预防和监督已经引起世界各国的广泛重视。现有的布鲁氏菌病诊断方法或多或少存在着操作复杂、耗时长、灵敏性低、易存在交叉反应等缺点。因此寻求操作简单、快速、灵敏的诊断方法,对于布鲁氏菌病早诊断、早治疗,减少其造成的医疗负担和经济损失具有非常重要的意义。本研究拟利用免疫信息学技术,对布鲁氏菌的主要外膜蛋白进行B细胞表位预测,然后结合融合蛋白技术,将布鲁氏菌外膜蛋白的优势抗原表位进行融合,制备出能够特异性识别布鲁氏菌抗体的融合蛋白,将其作为一种新型抗原。首先,融合蛋白作为诊断抗原,建立人布鲁氏菌病间接ELISA诊断方法;其次,融合蛋白与磁性纳米粒子相偶联,利用免疫磁珠分离技术制备可特异性识别布鲁氏菌抗体的磁性纳米生物探针,联合量子点与SPA偶联所制备的量子点荧光探针,建立一种布鲁氏菌病快速诊断技术。本课题的主要研究内容包括四个部分:第一部分布鲁氏菌融合蛋白的设计与评价。1通过查阅文献,经BLAST比对分析后,成功选取了4个抗原性良好的布鲁氏菌外膜蛋白:OMP31、OMP16、OMP2b和BP26;2利用免疫信息学参数(亲水性、柔性、表面可及性、β折叠)预测和线性B细胞表位预测工具,对选取的4个外膜蛋白进行预测,成功选取了15个重叠的B细胞表位;3将预测的15个B细胞表位通过linker肽链接后,成功构建了布鲁氏菌多表位融合蛋白,该蛋白共含440个氨基酸,分子量约60 KDa,对重组融合蛋白的抗原性进行预测,结果显示该融合蛋白抗原性良好。第二部分布鲁氏菌融合蛋白的表达、纯化和评价。1利用基因工程原理,根据自主设计的融合蛋白氨基酸序列,反推密码子,并进行密码子优化,优化成适合大肠杆菌表达的密码子,人工合成融合蛋白基因,成功合成了总长度为1338bp的融合蛋白基因,并成功连接到表达载体p ET-28b,构建重组质粒,将其成功转入感受态BL21(DE3),利用大肠杆菌表达系统进行融合蛋白表达,融合蛋白表达形式为包涵体蛋白,对包涵体进行变性和复性后,联合利用镍离子亲和层析和阴离子交换层析两种常用的蛋白纯化方法,对表达后的蛋白进行纯化,纯化后的蛋白浓度达1.217 mg/m L,SDS-PAGE凝胶电泳分析其纯度大于90%;2以融合蛋白为抗原,制备疫苗,通过免疫小鼠,成功诱导小鼠产生抗体,分析血清中抗体亚型主要为Ig G,通过流式细胞仪对小鼠脾细胞中T细胞亚型分析,CD4+和CD8+的百分比及CD4+/CD8+的比值均增加,说明融合蛋白诱导小鼠产生了较强的免疫反应,融合蛋白有较好的免疫原性;3通过间接ELISA方法,融合蛋白可以识别兔抗布鲁氏菌16M血清和鸡抗布鲁氏菌16M血清,表明融合蛋白有较好的抗原性,而融合蛋白不和兔抗大肠杆菌O157:H7血清和兔抗单核细胞增生性李斯特菌血清反应,表明该融合蛋白具有一定的特异性。第三部分人布鲁氏菌病间接ELISA诊断方法的建立和评价。1利用布鲁氏菌多表位重组蛋白作为诊断抗原,建立了一种布鲁氏菌病的间接ELISA诊断方法,并优化了该间接ELISA方法的一些反应条件,包括抗原包被浓度及包被时间、封闭液种类及封闭时间、一抗稀释度及一抗孵育时间、二抗稀释度及二抗孵育时间、底物显色时间等,并对优化后的间接ELISA方法的特异性、重复性、灵敏度等进行评价,结果良好;2利用优化后的间接ELISA方法对248份血清样本进行检测,吸光度值OD450做ROC曲线分析,结果曲线下面积为0.9409,Cut-off值为0.3865,灵敏性和特异性分别为88.89%和85.54%,阳性预测值和阴性预测值分别为93.75%和89.42%。与全菌抗原比较,该融合蛋白作为诊断抗原时,诊断的灵敏性、特异性等结果均优于全菌抗原。第四部分一种布鲁氏菌病快速诊断方法的建立及评价。1利用商品化纳米磁珠和荧光量子点,分别和布鲁氏菌多表位融合蛋白、SPA偶联,优化最佳偶联率,制备出了磁纳米探针和量子点荧光探针;2利用制备的两种生物探针,建立了一种新的布鲁氏菌病诊断方法,并对该方法的各种条件进行了优化,优化后的方法仅需要100 min就可以完成诊断,与间接ELISA方法比,明显缩短了诊断时间;3利用该方法对248份血清进行了诊断,测得的荧光强度做ROC曲线分析,结果显示该方法的曲线下面积为0.970,Cut-off值为150.4,灵敏性和特异性分别为96.15%和94.12%,阳性预测值和阴性预测值分别为:95.89%和94.12%,和间接ELISA方法比较,其灵敏性、特异性、阳性预测值和阴性预测值均优于间接ELISA,该方法的诊断效果较好。综上所述,本研究利用免疫信息学技术和融合蛋白技术,构建并表达了一种布鲁氏菌多表位融合蛋白,利用该融合蛋白建立了一种灵敏特异的人布鲁氏菌病间接ELISA诊断方法,结合免疫磁珠技术和量子点标记技术,制备了一种布鲁氏菌病快速诊断方法,对于开发具有我国自主知识产权的布鲁氏菌病诊断试剂盒具有重要意义。
[Abstract]:Brucellosis is a recurrent zoonosis. In recent years, the incidence of the disease has been increasing year by year, more than 500000 new cases in the world are added every year. The number of patients in China is increasing year by year. In 2015, 60000 cases of new cases are added. And it can cause great harm to human health. After human infection of brucellosis, patients will have fever, splenomegaly, bone and joint disease and hematological changes and other clinical symptoms, which can cause pregnant women to abort, neonatal death or birth weight loss. The diagnosis, prevention and supervision of brucellosis have caused the wide range of countries in the world. The existing diagnostic methods for brucellosis exist more or less the shortcomings of complex operation, long time, low sensitivity and easy cross reaction. Therefore, it is very important to seek simple, rapid and sensitive diagnostic methods for the early diagnosis and early treatment of brucellosis and reduce the medical burden and economic loss caused by brucellosis. This study intends to use immunological technology to predict the epitopes of the main outer membrane proteins of Brucella, and then combine the fusion protein technology to fuse the dominant epitopes of the outer membrane protein of Brucella, and prepare a fusion protein that can specifically identify the brucella antibody as a new type of antigen. The fusion protein is used as the diagnostic antigen to establish an indirect ELISA diagnosis method for human brucellosis. Secondly, the fusion protein is coupled with magnetic nanoparticles, and the magnetic nanoparticles are prepared by immunomagnetic bead separation technology to identify the specific identification of brucella antibody. The combined quantum dots and SPA coupling of quantum dots are used to establish the fluorescence probe. A rapid diagnostic technique for brucellosis. The main research contents of this topic include four parts: Part one, the design and evaluation of Brucella fusion protein.1, through consulting the literature, after BLAST comparison, successfully selected 4 Brucella outer membrane proteins with good antigenicity: OMP31, OMP16, OMP2b and BP26; 2 use immunological informatics. Parameters (hydrophilic, flexible, surface accessibility, beta folding) prediction and linear B cell epitope prediction tools were used to predict the selected 4 outer membrane proteins, and 15 overlapped B cell epitopes were successfully selected; 3 after linking the predicted 15 B cell epitopes through the linker peptide, a total of 440 Brucella fusion protein was constructed with a total of 440 The amino acid, molecular weight of about 60 KDa, predicted the antigenicity of the recombinant fusion protein. The results showed that the fusion protein was antigenicity. Second part of the fusion protein expression of Brucella, purified and evaluated the principle of.1 using gene engineering, according to the self designed fusion protein amino acid sequence, to reverse the cryptogram and optimize the codons. The fusion protein gene suitable for the expression of Escherichia coli was synthesized and the fusion protein gene was synthesized by artificial fusion protein gene. The fusion protein gene with a total length of 1338bp was successfully synthesized. The recombinant plasmid was successfully connected to the expression vector p ET-28b, and the recombinant plasmid was successfully transferred into the sensory BL21 (DE3). The fusion protein expression was expressed by the Escherichia coli expression system and the fusion protein table was used. After denaturation and refolding of inclusion body, two kinds of protein purification methods, nickel ion affinity chromatography and anion exchange chromatography, were used to purify the expressed protein, and the purified protein concentration was 1.217 mg/m L, and the purity of the purified protein was more than 90%, and 2 was resistant to fusion protein. In order to prepare the vaccine, the antibody was successfully induced in mice by immunizing mice. The antibody subtype was mainly Ig G in the serum. The percentage of T cells in the mouse spleen cells was analyzed by flow cytometry, the percentage of CD4+ and CD8+ and the ratio of CD4+/CD8+ were increased. It showed that the fusion egg white induced the stronger immune response and fusion protein in the mice. 3 the fusion protein can identify the Rabbit anti Brucella 16M serum and the chicken anti Brucella 16M serum by indirect ELISA method, indicating that the fusion protein has good antigenicity, and the fusion protein does not react with Rabbit anti Escherichia coli O157:H7 serum and Rabbit anti monocytic Lester sera serum reaction, indicating the fusion egg. White has certain specificity. The establishment and evaluation of the indirect ELISA diagnosis of brucellosis in third parts..1 uses the Brucella multi epitope protein as the diagnostic antigen. An indirect ELISA diagnosis method for brucellosis is established, and some reaction conditions of the indirect ELISA method are optimized, including the concentration of antigen inclusion and the concentration of the antigen. Time, type and closing time of closed liquid, one anti dilution and one anti incubation time, two anti dilution and two anti incubation time, substrate color time, etc., and evaluation of the specificity, repeatability and sensitivity of the optimized indirect ELISA method, and good results; 2 use the optimized indirect ELISA method to carry out 248 serum samples. The test and absorbance value OD450 were analyzed with ROC curve, the area under the curve was 0.9409, the Cut-off value was 0.3865, the sensitivity and specificity were 88.89% and 85.54% respectively. The positive predictive value and negative predictive value were 93.75% and 89.42%. respectively compared with the whole bacterial antigen. The fusion protein was the diagnostic sensitivity, specificity and so on as the diagnostic antigen. The fourth part of a rapid diagnosis method for brucellosis was established and evaluated by.1 using commercial nanomagnetic beads and fluorescence quantum dots, coupled with Brucella multi epitope fusion protein, SPA coupling, optimizing the optimum coupling rate, and preparing the magnetic nano probe and quantum dot fluorescence probe; 2 using two kinds of biological probes prepared. A new method for the diagnosis of brucellosis was established and the various conditions of the method were optimized. The optimized method was only 100 min to complete the diagnosis. Compared with the indirect ELISA method, the diagnosis time was shortened obviously. 3 the diagnosis of 248 sera was made by this method. The fluorescence intensity measured by the ROC curve was analyzed and the results showed a significant result. The area under the curve is 0.970, the Cut-off value is 150.4, the sensitivity and specificity are 96.15% and 94.12% respectively. The positive predictive value and negative predictive value are 95.89% and 94.12% respectively. Compared with the indirect ELISA method, the sensitivity, specificity, positive predictive value and negative predictive value are superior to those of indirect ELISA, and the diagnostic effect of this method is better. To sum up, a kind of Brucella multi epitope fusion protein was constructed and expressed by using immunological information technology and fusion protein technology. A sensitive and specific indirect ELISA diagnosis method for human brucellosis was established by using the fusion protein, and a kind of Brucella was prepared by combining immunomagnetic beads and quantum dot labeling technology. The rapid diagnosis method is of great significance for the development of brucellosis diagnostic kit with independent intellectual property rights in China.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R516.7

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