本文關(guān)鍵詞：羊布魯菌BCSP31蛋白的表達(dá)、純化及其單克隆抗體的制備和鑒定　出處：《第四軍醫(yī)大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 羊布魯菌 BCSP31 蛋白純化 單克隆抗體 ELISA

【摘要】： 布魯菌是引起人獸共患布魯菌病的病原菌。布魯菌有7個(gè)生物種型,其中羊、牛、豬三個(gè)種既可感染家畜也可以感染人類。布魯菌可通過多種途徑感染人和動(dòng)物,致病性強(qiáng),流行范圍廣,危害十分嚴(yán)重。此外,布魯菌還是一種潛在的生物戰(zhàn)劑。 布魯菌病臨床癥狀輕重不一,個(gè)體表現(xiàn)差異大,容易誤診,治療不當(dāng)會(huì)導(dǎo)致病情遷延,影響人畜健康并導(dǎo)致經(jīng)濟(jì)損失。此外,現(xiàn)有的布魯菌檢測(cè)方法仍不理想,存在特異性不高,不能區(qū)分疫苗免疫和自然感染,容易誤診和漏診。隨著近年來布魯菌病日益增高的流行趨勢(shì),發(fā)展快速敏感特異的檢測(cè)方法對(duì)控制布魯菌病疫情,及時(shí)處置病畜、治療患者有重要意義。 布魯菌細(xì)菌表面31kDa蛋白BCSP31具有抗原性強(qiáng)、在不同種間同源性高的特點(diǎn)。本研究擬通過純化BCSP31蛋白,制備其單克隆抗體(mAb),為建立布魯菌病ELISA檢測(cè)方法奠定基礎(chǔ)。 1主要方法 將質(zhì)粒pGEX-4T-1-BCSP31轉(zhuǎn)化至大腸桿菌并誘導(dǎo)表達(dá),采用GST親和層析純化的方法,分別獲得GST-BCSP31融合蛋白和BCSP31純化蛋白。用純化的BCSP31蛋白免疫小鼠,將小鼠脾細(xì)胞與骨髓瘤Sp2/0細(xì)胞融合,ELISA間接法篩選陽性克隆,3次克隆化后建立雜交瘤細(xì)胞系。采用小鼠腹腔接種雜交瘤細(xì)胞制備腹水,正辛酸-硫酸銨法純化mAb。采用Western-blot、ELISA以及表位相加試驗(yàn)等方法鑒定mAb特性。建立用于檢測(cè)布魯菌感染血清的ELISA間接夾心法。 2主要結(jié)果 GST-BCSP31融合蛋白在25℃誘導(dǎo)時(shí)為可溶性表達(dá),經(jīng)親和層析純化,分別獲得GST-BCSP31融合蛋白2.48 mg/mL和BCSP31純化蛋白0.5 mg/mL;Western-blot檢測(cè)結(jié)果顯示所獲蛋白可與兔抗布魯菌血清特異性反應(yīng)。獲得2株雜交瘤細(xì)胞系,1F1和1E5,均為IgG1亞類;2株mAb均可與羊布魯菌BCSP31蛋白特異性反應(yīng),而不與HCV AS3蛋白、結(jié)核分枝桿菌DAF 44a蛋白和RPF蛋白、漢坦病毒GP和NP以及人IgG1等無關(guān)蛋白反應(yīng),也不與大腸桿菌裂解液反應(yīng),表明這兩株mAb具有較高的特異性。并且這兩株mAb在BCSP31蛋白上的抗原位點(diǎn)不同;mAb 1F1與BCSP31蛋白的相對(duì)親和力高于1E5。ELISA間接夾心法的工作條件是以10μg/mL包被mAb,包被量為100μl/孔;抗原最佳濃度為20μg/mL,加入量為100μl/孔;HRP-羊抗兔最佳工作濃度為1:50000,加入量為100μl/孔。對(duì)相應(yīng)參數(shù)進(jìn)行評(píng)估,特異性、敏感性及重復(fù)性均良好。 BCSP31的純化及其mAb的獲得以及ELISA檢測(cè)方法的建立,不僅有助于進(jìn)一步了解與布魯菌感染和免疫相關(guān)的抗原成分及其作用機(jī)制,也為進(jìn)一步研制布魯菌檢測(cè)試劑盒提供了基礎(chǔ)。
[Abstract]:Brucella is causing pathogen zoonotic brucellosis. Brucella has 7 species, including sheep, cattle, three pigs can be infected animal can also infect humans. Infection through a variety of ways and animal Brucella, highly pathogenic, wide, harm is very serious. In addition, Brucella is a potential biological warfare agents.
Brucellosis is an important clinical symptoms, individual differences, easily misdiagnosed, improper treatment can lead to persistent disease, affect human and animal health and lead to economic losses. In addition, Brucella and the existing detection methods is still not ideal, specificity is not high, can not distinguish between natural infection and vaccination, misdiagnosis and missed with the popular trend in recent years. The diagnosis of brucellosis is increasing, the rapid development of sensitive and specific detection method to control the brucellosis epidemic, timely disposal of animal disease, it has important significance for treatment of patients.
Brucella bacterial surface 31kDa protein BCSP31 has strong antigenicity and high homology among different species. In this study, monoclonal antibody (mAb) was prepared by purification of BCSP31 protein, which lay the foundation for establishing ELISA detection method of brucellosis.
1 main methods
The plasmid was transformed into Escherichia coli pGEX-4T-1-BCSP31 and induced by GST affinity chromatography method, the obtained GST-BCSP31 fusion protein and BCSP31 protein were purified by BCSP31. The purified protein to immunize mouse, the mouse spleen cells and myeloma cell fusion of Sp2/0, the positive clones were screened by indirect ELISA, the establishment of hybridoma cell lines were cloned 3 times after the mice were inoculated with ascites. Preparation of hybridoma cells were positive by caprylic acid - ammonium sulfate purification of mAb. by Western-blot, ELISA and epitope additivity test method for the identification of mAb characteristics. Established for the detection of serum Shandong bacteria infection cloth ELISA indirect sandwich method.
2 main results
The expression of GST-BCSP31 fusion protein is soluble at 25 DEG C during induction, purified by affinity chromatography, respectively GST-BCSP31 fusion protein and purified BCSP31 protein 2.48 mg/mL 0.5 mg/mL; Western-blot showed that the bacteria protein serum specific reaction with Rabbit anti Brucella. 2 hybridoma cell lines, 1F1 and 1E5, are IgG1 sub class; 2 strains of mAb can be used with B.melitensis BCSP31 protein specific protein reaction, but not with HCV AS3, DAF of Mycobacterium tuberculosis 44a protein and RPF protein, Hantaan virus GP and NP and IgG1 related protein reaction does not react with Escherichia coli lysate, indicating the specificity of the two strains with mAb high. And the two strains of mAb antigenic sites on the BCSP31 protein of mAb and 1F1; the relative affinity of BCSP31 protein was higher than that of indirect sandwich 1E5.ELISA working conditions are based on 10 g/mL coated mAb, coating amount of 100 hole l/ antigen; The best concentration is 20 micron g/mL, the dosage is 100 micron l/ hole, the best working concentration of HRP- Goat anti rabbit is 1:50000, and the dosage is 100 HRP- l/ hole. The corresponding parameters are evaluated, the specificity, sensibility and reproducibility are good.
The purification of BCSP31, the acquisition of mAb and the establishment of ELISA detection method are not only helpful to further understand the antigen components and their action mechanisms associated with brucellosis and immunity, but also provide a basis for further developing Brucella detection kit.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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