结核分枝杆菌TB10.4蛋白单克隆抗体研制与鉴定

发布时间：2018-01-06 12:02

本文关键词：结核分枝杆菌TB10.4蛋白单克隆抗体研制与鉴定　出处：《扬州大学》2011年硕士论文　论文类型：学位论文

更多相关文章： 结核分枝杆菌 ESX TB10.4 单克隆抗体

【摘要】：长久以来人类做了不懈的努力去认清结核病漫长致病过程中发生的事件以及结核病原菌与宿主之间隐蔽复杂的关系,试图能在结核病防控困境中寻找新的突破口。结核病早期感染事件关连着宿主的先天性防御,影响着获得性免疫应答的方向。结核分枝杆早期分泌蛋白是感染早期事件中的重要效应分子,对它们的深入认识有助于为结核病的治疗、疫苗设计、早期诊断提供新的思路。存在于结核分枝杆菌早期分泌蛋白中的ESX家族成员是一群极有意思的小分子量蛋白,它们因显著的T细胞免疫原性倍受关注,曾是候选疫苗和诊断试剂热点研究靶区,其中极个别的蛋白已用于商业诊断或处于临床疫苗试验中；它们在疾病进程中作用重大但功能不明；它们之间相似的遗传特征及在G+中广泛存在性提示ESX家族蛋白具有普遍生物学意义；它们独特的分泌方式,代表着的G+新型分泌途径的T7S是近几年来揭起的极具竞争力的研究命题。所属ESX家族成员的TB10.4蛋白,是结核分枝杆菌生存必不可少的因子,并在致病过程中发挥重大作用,同时也是知之甚少的T7S/ESX-3系统的核心分泌蛋白。本课题采用原核表达不同标签的TB10.4融合蛋白产物作为单抗制备的免疫原和检测原,依照小鼠源性单克隆抗体经典制备流程筛选到8株稳定的TB10.4杂交瘤细胞系,其中有4株细胞株能与真核表达的TB10.4蛋白反应,这4株单抗有望为研究TB10.4分子在早期感染事件发挥的生物学功能、分子活动方式及T7S/ESX-3分泌机制功能提供有价值的工具。一.结核分枝杆菌TB10.4原核表达和鉴定构建携带两种融合标签的TB10.4原核表达重组载体pET-30(a)+-esxH和pGEX-6P-1-esH。提取测序正确的重组载体转化表达宿主菌BL21(DE3),对经抗生素选择培养出的重组BL21(DE3)(pET-30(a)+-esxH)BL21(DE3)(pGEX-6P-1-esxH)进行小量诱导表达；SDS-PAGE检测目的表达产物,对正确表达的重组菌蛋白表达条件进行优化；在蛋白表达优化条件下,大量诱导表达TB10.4融合蛋白,应用针对TB10.4融合标签的亲和层析柱纯化获得带6×His和GST标签的TB10.4蛋白,纯化的HIS-TB10.4和GST-TB10.4可分别作为TB10.4单克隆抗体研制的免疫原和检测原,从而为后续实验的开展提供了必要的生物材料。二.TB10.4单克隆抗体制备及特性鉴定以免疫原HIS-TB10.4免疫小鼠制取多抗血清,GST-TB10.4包被ELISA板棋盘滴定试验建立用于TB10.4单克隆抗体筛选的间接ELISA方法。单抗制备小鼠采用脾内免疫法,免疫原与弗氏完全佐剂乳化充分后,经皮下途径免疫BALB/c小鼠,每只100μg,21天后,进行脾内免疫,每只20μg,后三天取脾脏融合,依据经典小鼠源单克隆抗体程序,最终筛选出8株稳定分泌TB10.4单克隆抗体的杂交瘤细胞系。8株单抗均识别原核表达的TB10.4蛋白,表现出良好的特异性,其中的4株单抗能识别重组腺病毒表达的TB10.4蛋白。单抗亚型鉴定均为IgG1,制备的单抗腹水效价达到1×105-1×106的数量级。这些研制的单抗将在获取天然TB10.4蛋白,追踪TB10.4早期感染发生的分子事件,研究ESX-3分泌机制的构造成分及活动方式等方面发挥实际作用。
[Abstract]:For a long time people have made unremitting efforts to understand the relationship between the incidence of tuberculosis in the process of long pathogenic events and MTB and host hidden complex, trying to find a new breakthrough in the prevention and control of tuberculosis in trouble. Tuberculosis early infection event related with host innate defense, affect the acquired immune response in the direction of tuberculosis. Mycobacteria early secretory protein is an important effector molecule in the event of early infection, their understanding is helpful for the treatment of tuberculosis, vaccine design, early diagnosis and provide a new way of thinking. In Mycobacterium tuberculosis early secretory protein in the ESX family is a group of small molecular weight proteins very interesting, because they the primary T cells significantly attracted much attention, was a candidate vaccine and diagnostic reagent research target, which very few proteins have been used for commercial or diagnosis In the clinical trials; they are in the course of the disease is important but unknown function between them; similar genetic character and G+ in widespread existence suggests that ESX family proteins have a common biological significance; their unique way of representing the G+ secretion, secretion pathway of T7S is the new research topic in recent years uncovered very competitive. The members of the ESX family of TB10.4 protein is a factor essential to the survival of Mycobacterium tuberculosis, and play an important role in the pathogenic process, also a core knowledge T7S/ESX-3 system about the secretory protein.
The expression of different immune label by prokaryotic TB10.4 fusion protein as the original monoclonal antibody preparation and detection of the original, in accordance with the mouse monoclonal antibody to TB10.4 classic preparation process of screening 8 strains of hybridoma cell lines stably, including TB10.4 protein reaction 4 cell lines can and eukaryotic expression of the 4. The monoclonal antibody is expected to play the biological function of TB10.4 molecule in the early infection event, molecular activity and T7S/ESX-3 secretion function provide valuable tools.
TB10.4 prokaryotic expression and identification of Mycobacterium tuberculosis
To construct two fusion tag TB10.4 Recombinant Prokaryotic expression vector pET-30 (a) +-esxH and pGEX-6P-1-esH. to extract the correct sequencing recombinant plasmid transformed into host strain BL21 (DE3), on the choice of antibiotics produce recombinant BL21 (DE3) (pET-30 (a) +-esxH) BL21 (DE3) (pGEX-6P-1-esxH) of induction objective to detect SDS-PAGE expression; the expression product, to optimize the expression conditions of recombinant protein was correctly expressed in protein expression; under the optimum conditions, a large number of inducible expression of TB10.4 fusion protein, application for His and GST with 6 * TB10.4 tag protein purification TB10.4 fusion tag affinity chromatography, purified HIS-TB10.4 and GST-TB10.4 respectively as TB10.4 developed the original immune monoclonal antibody and detection of the original, so as to provide the necessary materials for further biological experiments.
Preparation and characterization of two.TB10.4 monoclonal antibody
In immunized mice immunized with HIS-TB10.4 polyclonal antibody preparation, GST-TB10.4 coated ELISA plate board titration test established for screening TB10.4 monoclonal antibody indirect ELISA method. The preparation of monoclonal antibody in mice by intrasplenic immunization, immunogen emulsified with complete Freund's adjuvant fully after percutaneous approach to immune BALB/c mice, each 100 g. 21 days after intrasplenic immunization, each 20 g, three days after the spleen fusion, based on classical mouse monoclonal antibody, finally screened 8 strains of hybridoma cell lines secreting monoclonal antibodies against TB10.4.8 mAbs recognize prokaryotic expression of TB10.4 protein showed good specificity. One of the 4 mAbs can identify the expression of recombinant adenovirus TB10.4 protein monoclonal antibody subtypes were identified as IgG1 monoclonal antibody, titer of ascites preparation reached 1 * 105-1 * 106 magnitude.
These McAbs will play a practical role in obtaining natural TB10.4 protein, tracing the molecular events of early infection of TB10.4, studying the composition and activity mode of ESX-3 secretion mechanism.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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