本文選題：布魯氏菌病 + 融合蛋白��； 參考：《吉林大學(xué)》2016年博士論文

【摘要】：布魯氏菌病是一種再發(fā)的人獸共患傳染病,近年來發(fā)病率呈逐年上升的趨勢(shì),全球每年超過500 000新增病例,我國(guó)發(fā)病人數(shù)也在逐年增長(zhǎng),2015年全國(guó)新增病例60 000例。布魯氏菌病不僅會(huì)造成感染動(dòng)物流產(chǎn),給畜牧業(yè)帶來巨大經(jīng)濟(jì)損失,而且會(huì)給人類的健康造成巨大危害。人類感染布魯氏菌病后,患者會(huì)出現(xiàn)發(fā)熱、肝脾腫大、骨關(guān)節(jié)病及血液學(xué)改變等臨床癥狀,嚴(yán)重時(shí)會(huì)造成孕婦流產(chǎn)、新生兒死亡或者出生體重降低。對(duì)布魯氏菌病的診斷、預(yù)防和監(jiān)督已經(jīng)引起世界各國(guó)的廣泛重視。現(xiàn)有的布魯氏菌病診斷方法或多或少存在著操作復(fù)雜、耗時(shí)長(zhǎng)、靈敏性低、易存在交叉反應(yīng)等缺點(diǎn)。因此尋求操作簡(jiǎn)單、快速、靈敏的診斷方法,對(duì)于布魯氏菌病早診斷、早治療,減少其造成的醫(yī)療負(fù)擔(dān)和經(jīng)濟(jì)損失具有非常重要的意義。本研究擬利用免疫信息學(xué)技術(shù),對(duì)布魯氏菌的主要外膜蛋白進(jìn)行B細(xì)胞表位預(yù)測(cè),然后結(jié)合融合蛋白技術(shù),將布魯氏菌外膜蛋白的優(yōu)勢(shì)抗原表位進(jìn)行融合,制備出能夠特異性識(shí)別布魯氏菌抗體的融合蛋白,將其作為一種新型抗原。首先,融合蛋白作為診斷抗原,建立人布魯氏菌病間接ELISA診斷方法;其次,融合蛋白與磁性納米粒子相偶聯(lián),利用免疫磁珠分離技術(shù)制備可特異性識(shí)別布魯氏菌抗體的磁性納米生物探針,聯(lián)合量子點(diǎn)與SPA偶聯(lián)所制備的量子點(diǎn)熒光探針,建立一種布魯氏菌病快速診斷技術(shù)。本課題的主要研究?jī)?nèi)容包括四個(gè)部分:第一部分布魯氏菌融合蛋白的設(shè)計(jì)與評(píng)價(jià)。1通過查閱文獻(xiàn),經(jīng)BLAST比對(duì)分析后,成功選取了4個(gè)抗原性良好的布魯氏菌外膜蛋白:OMP31、OMP16、OMP2b和BP26;2利用免疫信息學(xué)參數(shù)(親水性、柔性、表面可及性、β折疊)預(yù)測(cè)和線性B細(xì)胞表位預(yù)測(cè)工具,對(duì)選取的4個(gè)外膜蛋白進(jìn)行預(yù)測(cè),成功選取了15個(gè)重疊的B細(xì)胞表位;3將預(yù)測(cè)的15個(gè)B細(xì)胞表位通過linker肽鏈接后,成功構(gòu)建了布魯氏菌多表位融合蛋白,該蛋白共含440個(gè)氨基酸,分子量約60 KDa,對(duì)重組融合蛋白的抗原性進(jìn)行預(yù)測(cè),結(jié)果顯示該融合蛋白抗原性良好。第二部分布魯氏菌融合蛋白的表達(dá)、純化和評(píng)價(jià)。1利用基因工程原理,根據(jù)自主設(shè)計(jì)的融合蛋白氨基酸序列,反推密碼子,并進(jìn)行密碼子優(yōu)化,優(yōu)化成適合大腸桿菌表達(dá)的密碼子,人工合成融合蛋白基因,成功合成了總長(zhǎng)度為1338bp的融合蛋白基因,并成功連接到表達(dá)載體p ET-28b,構(gòu)建重組質(zhì)粒,將其成功轉(zhuǎn)入感受態(tài)BL21(DE3),利用大腸桿菌表達(dá)系統(tǒng)進(jìn)行融合蛋白表達(dá),融合蛋白表達(dá)形式為包涵體蛋白,對(duì)包涵體進(jìn)行變性和復(fù)性后,聯(lián)合利用鎳離子親和層析和陰離子交換層析兩種常用的蛋白純化方法,對(duì)表達(dá)后的蛋白進(jìn)行純化,純化后的蛋白濃度達(dá)1.217 mg/m L,SDS-PAGE凝膠電泳分析其純度大于90%;2以融合蛋白為抗原,制備疫苗,通過免疫小鼠,成功誘導(dǎo)小鼠產(chǎn)生抗體,分析血清中抗體亞型主要為Ig G,通過流式細(xì)胞儀對(duì)小鼠脾細(xì)胞中T細(xì)胞亞型分析,CD4+和CD8+的百分比及CD4+/CD8+的比值均增加,說明融合蛋白誘導(dǎo)小鼠產(chǎn)生了較強(qiáng)的免疫反應(yīng),融合蛋白有較好的免疫原性;3通過間接ELISA方法,融合蛋白可以識(shí)別兔抗布魯氏菌16M血清和雞抗布魯氏菌16M血清,表明融合蛋白有較好的抗原性,而融合蛋白不和兔抗大腸桿菌O157:H7血清和兔抗單核細(xì)胞增生性李斯特菌血清反應(yīng),表明該融合蛋白具有一定的特異性。第三部分人布魯氏菌病間接ELISA診斷方法的建立和評(píng)價(jià)。1利用布魯氏菌多表位重組蛋白作為診斷抗原,建立了一種布魯氏菌病的間接ELISA診斷方法,并優(yōu)化了該間接ELISA方法的一些反應(yīng)條件,包括抗原包被濃度及包被時(shí)間、封閉液種類及封閉時(shí)間、一抗稀釋度及一抗孵育時(shí)間、二抗稀釋度及二抗孵育時(shí)間、底物顯色時(shí)間等,并對(duì)優(yōu)化后的間接ELISA方法的特異性、重復(fù)性、靈敏度等進(jìn)行評(píng)價(jià),結(jié)果良好;2利用優(yōu)化后的間接ELISA方法對(duì)248份血清樣本進(jìn)行檢測(cè),吸光度值OD450做ROC曲線分析,結(jié)果曲線下面積為0.9409,Cut-off值為0.3865,靈敏性和特異性分別為88.89%和85.54%,陽(yáng)性預(yù)測(cè)值和陰性預(yù)測(cè)值分別為93.75%和89.42%。與全菌抗原比較,該融合蛋白作為診斷抗原時(shí),診斷的靈敏性、特異性等結(jié)果均優(yōu)于全菌抗原。第四部分一種布魯氏菌病快速診斷方法的建立及評(píng)價(jià)。1利用商品化納米磁珠和熒光量子點(diǎn),分別和布魯氏菌多表位融合蛋白、SPA偶聯(lián),優(yōu)化最佳偶聯(lián)率,制備出了磁納米探針和量子點(diǎn)熒光探針;2利用制備的兩種生物探針,建立了一種新的布魯氏菌病診斷方法,并對(duì)該方法的各種條件進(jìn)行了優(yōu)化,優(yōu)化后的方法僅需要100 min就可以完成診斷,與間接ELISA方法比,明顯縮短了診斷時(shí)間;3利用該方法對(duì)248份血清進(jìn)行了診斷,測(cè)得的熒光強(qiáng)度做ROC曲線分析,結(jié)果顯示該方法的曲線下面積為0.970,Cut-off值為150.4,靈敏性和特異性分別為96.15%和94.12%,陽(yáng)性預(yù)測(cè)值和陰性預(yù)測(cè)值分別為:95.89%和94.12%,和間接ELISA方法比較,其靈敏性、特異性、陽(yáng)性預(yù)測(cè)值和陰性預(yù)測(cè)值均優(yōu)于間接ELISA,該方法的診斷效果較好。綜上所述,本研究利用免疫信息學(xué)技術(shù)和融合蛋白技術(shù),構(gòu)建并表達(dá)了一種布魯氏菌多表位融合蛋白,利用該融合蛋白建立了一種靈敏特異的人布魯氏菌病間接ELISA診斷方法,結(jié)合免疫磁珠技術(shù)和量子點(diǎn)標(biāo)記技術(shù),制備了一種布魯氏菌病快速診斷方法,對(duì)于開發(fā)具有我國(guó)自主知識(shí)產(chǎn)權(quán)的布魯氏菌病診斷試劑盒具有重要意義。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R516.7

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