發(fā)布時(shí)間：2018-03-16 15:35

  本文選題：布魯氏菌　切入點(diǎn)：多克隆抗體　出處：《吉林大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：布魯氏菌是一種重要的人獸共患病病原菌,它的傳播和流行給畜牧業(yè)及人類(lèi)健康造成巨大的威脅,加強(qiáng)對(duì)其檢測(cè),對(duì)于布魯氏菌病的防控具有重要的意義�，F(xiàn)有的布魯氏菌檢測(cè)方法或多或少存在著操作復(fù)雜、耗時(shí)較長(zhǎng)、靈敏度低以及特異性差等缺點(diǎn),因此建立快速、靈敏且特異的布魯氏菌檢測(cè)方法十分必要。本課題旨在制備布魯氏菌兔多克隆抗體,并聯(lián)合免疫磁珠分離技術(shù)和量子點(diǎn)標(biāo)記技術(shù)建立一種布魯氏菌快速檢測(cè)方法,并對(duì)該方法的檢測(cè)效果進(jìn)行評(píng)價(jià)。本課題的研究?jī)?nèi)容包括以下兩個(gè)方面:1.布魯氏菌兔多克隆抗體的制備及鑒定采用布魯氏菌16M制備疫苗,免疫新西蘭大白兔,制備多克隆抗體,利用飽和硫酸銨鹽析法對(duì)抗體進(jìn)行純化。建立間接ELISA方法,使用矩陣法對(duì)以下反應(yīng)條件進(jìn)行優(yōu)化,包括抗原包被濃度、血清工作濃度、包被條件、封閉液種類(lèi)、封閉時(shí)間、抗原抗體反應(yīng)時(shí)間、二抗工作濃度、二抗反應(yīng)時(shí)間和顯色時(shí)間,并用優(yōu)化的間接ELISA方法測(cè)定抗體效價(jià)和其特異性;采用SDS-PAGE方法測(cè)定多克隆抗體的純度;采用Bradford蛋白濃度測(cè)定試劑盒測(cè)定多克隆抗體的蛋白含量。結(jié)果顯示,間接ELISA方法的最優(yōu)條件為:最佳抗原包被條件為37℃包被1 h、濃度為1:40,最佳封閉液為5%脫脂奶粉,最佳封閉時(shí)間為1 h,最佳血清工作濃度為1:16000,最佳抗原抗體反應(yīng)時(shí)間為1.5 h,最佳二抗工作濃度為1:10000,二抗反應(yīng)時(shí)間為0.5 h,最佳顯色時(shí)間為10 min;1號(hào)抗體效價(jià)為1:256000,2號(hào)抗體效價(jià)為1:2048000;抗體特異性較好,與大腸桿菌O157、單增李斯特菌、奇異變形桿菌、陰溝腸桿菌、坂崎腸桿菌、克雷伯桿菌、鮑氏志賀氏菌、金黃色葡萄球菌、粘質(zhì)沙雷氏菌、沙門(mén)氏菌交叉反應(yīng)小;抗體純度較好;1號(hào)和2號(hào)抗體蛋白含量分別為14.091 mg/m L和17.454 mg/m L。2.檢測(cè)方法的建立與評(píng)價(jià)將羧基化磁珠與制備的布魯氏菌多克隆抗體共價(jià)偶聯(lián),制備可識(shí)別布魯氏菌的磁納米探針;將羧基化熒光量子點(diǎn)與實(shí)驗(yàn)室前期制備的布魯氏菌雞卵黃抗體(Ig Y)共價(jià)偶聯(lián),制備可以識(shí)別布魯氏菌的量子點(diǎn)熒光生物探針,基于這兩種生物探針建立一種新的布魯氏菌檢測(cè)方法,得到磁納米探針—布魯氏菌—量子點(diǎn)熒光生物探針“三明治”夾心復(fù)合物,利用熒光光譜儀對(duì)復(fù)合物進(jìn)行熒光檢測(cè),建立熒光強(qiáng)度與菌液濃度的線性回歸方程,從而實(shí)現(xiàn)對(duì)布魯氏菌的快速、定量的檢測(cè)。在檢測(cè)方法建立過(guò)程中,對(duì)磁納米探針的用量、富集時(shí)間、量子點(diǎn)探針的用量以及標(biāo)記時(shí)間進(jìn)行優(yōu)化,評(píng)價(jià)檢測(cè)方法的靈敏度、特異性和重復(fù)性,并用所建立的檢測(cè)方法對(duì)人工模擬的肉樣和牛奶樣品進(jìn)行檢測(cè)。結(jié)果顯示,磁納米生物探針制備的最佳條件為100μL磁珠與122.178μg兔多克隆抗體于37℃偶聯(lián)2 h;量子點(diǎn)生物探針制備的最佳條件為10μL QDs與262μg Ig Y于37℃偶聯(lián)2 h。所建立的布魯氏菌檢測(cè)方法最佳反應(yīng)條件為:磁納米探針最佳用量為100μL,最佳富集時(shí)間為45 min,量子點(diǎn)探針最佳用量為500μL,最佳標(biāo)記時(shí)間為60 min;該方法的檢測(cè)時(shí)間約為105 min,反應(yīng)產(chǎn)物的熒光強(qiáng)度與菌液濃度的對(duì)數(shù)呈線性關(guān)系,線性方程為y=21.763x+78.371(R2=0.9983),檢測(cè)限為103 CFU/m L;特異性實(shí)驗(yàn)結(jié)果顯示,該方法能識(shí)別羊種和豬種布魯氏菌,但是不能識(shí)別大腸桿菌O157和單增李斯特菌,特異性較好;在牛奶模擬樣和肉汁模擬樣中的檢測(cè)限均為102 CFU/m L,說(shuō)明該方法在模擬樣品中的檢測(cè)效果較好。綜上所述,本課題制備了布魯氏菌兔多克隆抗體、能夠識(shí)別布魯氏菌的磁納米生物探針和量子點(diǎn)熒光生物探針,并建立了一種基于磁性分離技術(shù)和量子點(diǎn)標(biāo)記技術(shù)的布魯氏菌快速檢測(cè)方法。該檢測(cè)方法的檢測(cè)限較低,特異性較好,且具有較好的穩(wěn)定性,在模擬樣品的檢測(cè)中也能得到良好的應(yīng)用,本研究工作建立了具有我國(guó)自主知識(shí)產(chǎn)權(quán)的布魯氏菌的快速檢測(cè)方法。
[Abstract]:Brucellosis is an important zoonotic pathogen, its spread and epidemic caused a great threat to human health and animal husbandry, strengthen the inspection, has an important significance for the prevention and control of brucellosis Brucella. The existing detection methods are more or less complicated operation, time-consuming, low sensitivity and poor specificity shortcomings, so establishing a rapid detection method, it is necessary to Brucella sensitive and specific. This paper aims at the preparation of Brucella polyclonal antibody, combined with immunomagnetic separation technique and quantum dot marking technology to establish a rapid detection of Brucella, and evaluate the effect of the detection method. The content of this research include the following two aspects: preparation and identification of Brucella using 1. rabbit polyclonal antibody of Brucella 16M vaccine, immunization of New Zealand white rabbits, preparation The polyclonal antibody, the antibody was purified by saturated ammonium sulfate salting out method. The establishment of indirect ELISA method, using matrix method to optimize the reaction conditions, including antigen concentration, serum concentration, coating condition, blocking solution, blocking time, the antigen antibody reaction time, concentration of two two anti, anti reaction time and time of color, determination of antibody titer and specificity and by indirect ELISA optimization method; Determination of purity of polyclonal antibody by SDS-PAGE method; Determination of protein assay kit using polyclonal antibody against Bradford protein concentration. The results showed that the optimal conditions of indirect ELISA method for antigen: the best conditions for the 37 C was 1 h, the concentration of the sealed liquid for 1:40, 5% skim milk powder, the best blocking time is 1 h, the concentration of serum 1:16000 for the best work, the best antigen antibody reaction time is 1.5 h, the best anti two The concentration of 1:10000, two anti reaction time is 0.5 h, the best color time for 10 min; 1, the antibody titer was 1:256000,2, antibody titer of 1:2048000 antibody; specificity, O157 and Escherichia coli, Listeria bacteria Lester, Proteus mirabilis, Enterobacter cloacae, Enterobacter sakazakii, Klebsiella pneumoniae Bao Shizhi coli, Shigella, Staphylococcus aureus, Serratia marcescens, Salmonella antibody cross reactivity; high purity; No. 1 and No. 2 antibody protein content respectively for the establishment and evaluation of 14.091 mg/m L and 17.454 mg/m L.2. detection method to the carboxylic beads and preparation of Brucella polyclonal antibody covalently coupling, preparation of magnetic nano probes for identification of Brucella; carboxylated Brucella egg yolk antibody pre preparation of fluorescent quantum dots and laboratory (Ig Y) covalent coupling, preparation of quantum dot fluorescent probe identification of brucella, These two kinds of biological probes to establish a new method based on the detection of Brucella, magnetic nano probe - Quantum Dots Fluorescent Probe of Brucella sandwich complexes, fluorescence detection of complexes by fluorescence spectrometer, a linear fluorescence intensity and the concentration of the bacteria liquid regression equation, thus realizing the Brucella fast. Quantitative detection. In the process of establishing a detection method, the magnetic nano probe dosage, preconcentration time, dosage of quantum dots and mark time optimization, sensitivity evaluation method, specificity and reproducibility, and the method of artificial meat and milk samples were detected. The results showed the best conditions, magnetic nano bio probe preparation for 100 L and 122.178 G using rabbit polyclonal antibody of 37 DEG C to 2 h coupling; quantum optimal point biological probe preparation Brucella detection method of the optimum reaction conditions for 10 L QDs and 262 g Ig 2 h. Y to 37 DEG C coupling established: magnetic nano probes the best dosage is 100 L, the best enrichment time is 45 min, QD probes the best dosage is 500 L, the best time is 60 min marker; detection this method is about 105 min, there was a linear relationship between the logarithm of the fluorescence intensity and the concentration of bacterium of reaction product, linear equation y=21.763x+78.371 (R2=0.9983), the detection limit is 103 CFU/m L; specific experimental results show that the method can identify the sheep and swine Brucella, but not the identification of Escherichia coli O157 and Listeria by Lester bacteria, better specificity; in milk samples and samples of simulation simulation gravy detection limit was 102 CFU/m L, to improve the detection effect in simulated samples. In summary, the preparation of Brucella polyclonal antibody, which can be identified The magnetic nano bio probe and quantum dot fluorescent probe not of Brucella, and establish a method for rapid detection of magnetic separation technique and quantum dot labeling technique based on the detection of Brucella. The detection limit is low, and has good specificity, good stability, can also be a good application in the detection of simulated samples in this work, a rapid detection method with our own intellectual property rights. Brucella

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R155

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