發(fā)布時(shí)間：2018-01-06 12:02

  本文關(guān)鍵詞：結(jié)核分枝桿菌TB10.4蛋白單克隆抗體研制與鑒定　出處：《揚(yáng)州大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 結(jié)核分枝桿菌 ESX TB10.4 單克隆抗體

【摘要】：長(zhǎng)久以來(lái)人類(lèi)做了不懈的努力去認(rèn)清結(jié)核病漫長(zhǎng)致病過(guò)程中發(fā)生的事件以及結(jié)核病原菌與宿主之間隱蔽復(fù)雜的關(guān)系,試圖能在結(jié)核病防控困境中尋找新的突破口。結(jié)核病早期感染事件關(guān)連著宿主的先天性防御,影響著獲得性免疫應(yīng)答的方向。結(jié)核分枝桿早期分泌蛋白是感染早期事件中的重要效應(yīng)分子,對(duì)它們的深入認(rèn)識(shí)有助于為結(jié)核病的治療、疫苗設(shè)計(jì)、早期診斷提供新的思路。存在于結(jié)核分枝桿菌早期分泌蛋白中的ESX家族成員是一群極有意思的小分子量蛋白,它們因顯著的T細(xì)胞免疫原性倍受關(guān)注,曾是候選疫苗和診斷試劑熱點(diǎn)研究靶區(qū),其中極個(gè)別的蛋白已用于商業(yè)診斷或處于臨床疫苗試驗(yàn)中；它們?cè)诩膊∵M(jìn)程中作用重大但功能不明；它們之間相似的遺傳特征及在G+中廣泛存在性提示ESX家族蛋白具有普遍生物學(xué)意義；它們獨(dú)特的分泌方式,代表著的G+新型分泌途徑的T7S是近幾年來(lái)揭起的極具競(jìng)爭(zhēng)力的研究命題。所屬ESX家族成員的TB10.4蛋白,是結(jié)核分枝桿菌生存必不可少的因子,并在致病過(guò)程中發(fā)揮重大作用,同時(shí)也是知之甚少的T7S/ESX-3系統(tǒng)的核心分泌蛋白。 本課題采用原核表達(dá)不同標(biāo)簽的TB10.4融合蛋白產(chǎn)物作為單抗制備的免疫原和檢測(cè)原,依照小鼠源性單克隆抗體經(jīng)典制備流程篩選到8株穩(wěn)定的TB10.4雜交瘤細(xì)胞系,其中有4株細(xì)胞株能與真核表達(dá)的TB10.4蛋白反應(yīng),這4株單抗有望為研究TB10.4分子在早期感染事件發(fā)揮的生物學(xué)功能、分子活動(dòng)方式及T7S/ESX-3分泌機(jī)制功能提供有價(jià)值的工具。 一.結(jié)核分枝桿菌TB10.4原核表達(dá)和鑒定 構(gòu)建攜帶兩種融合標(biāo)簽的TB10.4原核表達(dá)重組載體pET-30(a)+-esxH和pGEX-6P-1-esH。提取測(cè)序正確的重組載體轉(zhuǎn)化表達(dá)宿主菌BL21(DE3),對(duì)經(jīng)抗生素選擇培養(yǎng)出的重組BL21(DE3)(pET-30(a)+-esxH)BL21(DE3)(pGEX-6P-1-esxH)進(jìn)行小量誘導(dǎo)表達(dá)；SDS-PAGE檢測(cè)目的表達(dá)產(chǎn)物,對(duì)正確表達(dá)的重組菌蛋白表達(dá)條件進(jìn)行優(yōu)化；在蛋白表達(dá)優(yōu)化條件下,大量誘導(dǎo)表達(dá)TB10.4融合蛋白,應(yīng)用針對(duì)TB10.4融合標(biāo)簽的親和層析柱純化獲得帶6×His和GST標(biāo)簽的TB10.4蛋白,純化的HIS-TB10.4和GST-TB10.4可分別作為T(mén)B10.4單克隆抗體研制的免疫原和檢測(cè)原,從而為后續(xù)實(shí)驗(yàn)的開(kāi)展提供了必要的生物材料。 二.TB10.4單克隆抗體制備及特性鑒定 以免疫原HIS-TB10.4免疫小鼠制取多抗血清,GST-TB10.4包被ELISA板棋盤(pán)滴定試驗(yàn)建立用于TB10.4單克隆抗體篩選的間接ELISA方法。單抗制備小鼠采用脾內(nèi)免疫法,免疫原與弗氏完全佐劑乳化充分后,經(jīng)皮下途徑免疫BALB/c小鼠,每只100μg,21天后,進(jìn)行脾內(nèi)免疫,每只20μg,后三天取脾臟融合,依據(jù)經(jīng)典小鼠源單克隆抗體程序,最終篩選出8株穩(wěn)定分泌TB10.4單克隆抗體的雜交瘤細(xì)胞系。8株單抗均識(shí)別原核表達(dá)的TB10.4蛋白,表現(xiàn)出良好的特異性,其中的4株單抗能識(shí)別重組腺病毒表達(dá)的TB10.4蛋白。單抗亞型鑒定均為IgG1,制備的單抗腹水效價(jià)達(dá)到1×105-1×106的數(shù)量級(jí)。 這些研制的單抗將在獲取天然TB10.4蛋白,追蹤TB10.4早期感染發(fā)生的分子事件,研究ESX-3分泌機(jī)制的構(gòu)造成分及活動(dòng)方式等方面發(fā)揮實(shí)際作用。
[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.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392

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