發(fā)布時間：2018-06-06 14:04

  本文選題：結(jié)核分枝桿菌 + 基因疫苗��； 參考：《四川大學(xué)》2007年博士論文

【摘要】： 結(jié)核病是人類主要的傳染性疾病之一。全球每年新發(fā)結(jié)核病人數(shù)超過800萬，死亡人數(shù)約300萬，全世界1／3人口受到結(jié)核菌感染。近年來HIV的廣泛流行及多重耐藥結(jié)核菌株的出現(xiàn)使結(jié)核病的發(fā)病率和死亡率呈上升趨勢。我國結(jié)核病疫情在全球?qū)俑吡餍械貐^(qū)，結(jié)核病人數(shù)居全世界第二位，是全球22個結(jié)核病高負(fù)擔(dān)國家之一。據(jù)國家衛(wèi)生部2006年3月24日的報告，結(jié)核病的發(fā)病率和死亡率都居于傳染病首位。世界衛(wèi)生組織早在1993年宣布“全球結(jié)核病緊急狀態(tài)”，并指出應(yīng)優(yōu)先發(fā)展有效的結(jié)核病疫苗。 目前用于預(yù)防結(jié)核病的唯一疫苗是卡介苗(BCG)，它是牛分枝桿菌的減毒活菌苗株。由于反復(fù)傳代的原因，卡介苗一些重要的免疫保護(hù)性抗原基因已發(fā)生變異，免疫保護(hù)效果也大不如前。免疫保護(hù)力在不同人群中差異極大，為0-80％；通過連續(xù)免疫接種BCG企圖降低肺結(jié)核發(fā)生率的實(shí)驗(yàn)結(jié)果也令人失望，因此，改造BCG、開發(fā)新疫苗已成為研究熱點(diǎn)。目前結(jié)核病疫苗主要有：基因工程重組BCG、亞單位疫苗、基因疫苗、營養(yǎng)缺陷型疫苗等類型。 白細(xì)胞介素12(IL-12)是由單核細(xì)胞、巨噬細(xì)胞分泌產(chǎn)生的一種細(xì)胞因子，具有廣泛的生物學(xué)活性，是連接天然免疫和獲得性免疫的功能性橋梁，是目前發(fā)現(xiàn)對體內(nèi)免疫活性細(xì)胞誘導(dǎo)和調(diào)節(jié)作用最強(qiáng)、范圍最廣的細(xì)胞因子；在誘導(dǎo)Th0細(xì)胞向Th1型細(xì)胞發(fā)育過程中發(fā)揮重要作用，可明顯增強(qiáng)細(xì)胞免疫功能。 本研究報道了IL-12真核表達(dá)質(zhì)粒及IL-12基因重組卡介苗的構(gòu)建，并對IL-12與結(jié)核桿菌免疫保護(hù)性抗原基因疫苗聯(lián)合免疫及IL-12基因重組卡介苗的免疫原性以及在調(diào)節(jié)機(jī)體免疫應(yīng)答方面的作用進(jìn)行了研究。IL-12在正常情況下為非組成性表達(dá)，我們首先經(jīng)體外分別誘導(dǎo)人白血病細(xì)胞株THP-1和HL-60，得到IL-12 p40、p35及p40-p35融合基因。將IL-12基因克隆入真核表達(dá)質(zhì)粒pcDNA3.1(+)／myc-His中，獲得IL-12基因真核表達(dá)質(zhì)粒pcDNA-IL-12，并將其轉(zhuǎn)染COS-7細(xì)胞，通過RT-PCR及ELISA檢測進(jìn)行了鑒定。ELISA檢測證實(shí)轉(zhuǎn)染后的COS-7細(xì)胞能分泌表達(dá)IL-12。 為研究細(xì)胞因子和結(jié)核菌保護(hù)性抗原基因疫苗共同免疫后，對小鼠體內(nèi)免疫應(yīng)答的誘導(dǎo)及小鼠免疫功能的影響，我們將IL-12和ESAT-6抗原真核表達(dá)質(zhì)粒共同肌肉免疫BALB／c小鼠，結(jié)果發(fā)現(xiàn)IL-12與ESAT-6基因聯(lián)合免疫，較單獨(dú)的ESAT-6基因免疫，刺激機(jī)體產(chǎn)生更加強(qiáng)烈而穩(wěn)定的細(xì)胞免疫應(yīng)答。IL-12與ESAT-6聯(lián)合免疫，ESAT-6使用劑量是ESAT-6單獨(dú)免疫時的一半，但是誘導(dǎo)的免疫反應(yīng)卻明顯強(qiáng)于ESAT-6單獨(dú)免疫，而與BCG免疫相當(dāng)。 另外，我們將IL-12基因連接到pMV361載體上，構(gòu)建含人IL-12基因的重組大腸桿菌—分枝桿菌穿梭質(zhì)粒rpMV-12，，通過電穿孔的方法轉(zhuǎn)化BCG。該基因重組BCG經(jīng)卡那霉素抗性篩選和PCR擴(kuò)增鑒定，進(jìn)一步通過重組BCG誘導(dǎo)表達(dá)后的SDS-PAGE電泳檢測，篩選得到能分泌表達(dá)IL-12基因的重組BCG菌株rBCG-12。 接著我們用IL-12基因重組BCG(rBCG-12)和傳統(tǒng)BGG分別免疫小鼠，觀察免疫后不同時間點(diǎn)小鼠體內(nèi)細(xì)胞免疫和體液免疫的變化。將rBCG-12和BCG皮下接種BALB／c小鼠(10~6 CFU)，分別在免疫接種后第6、8、10、12周時，測定血清特異性抗體水平，脾淋巴細(xì)胞增殖能力，IFN-γ和IL-4分泌量。實(shí)驗(yàn)結(jié)果顯示，rBCG-12免疫組的脾淋巴細(xì)胞增殖能力、IFN-γ分泌水平均明顯高于對照組和BCG免疫組，并且維持的時間也更長。而rBCG-12免疫組、BCG免疫組和對照組血清特異性抗體水平和IL-4分泌水平差異沒有統(tǒng)計(jì)學(xué)意義。
[Abstract]:Tuberculosis is one of the major infectious diseases in the world . The number of new TB patients worldwide is over 8 million , the number of deaths is about 3 million , and 1 / 3 of the world ' s population is affected by TB . In recent years , the prevalence of tuberculosis and the emergence of multiple - resistant TB strains have caused tuberculosis infection . In recent years , the incidence and mortality of tuberculosis have been the first in the world . According to the report of the Ministry of Health of the Ministry of Health on March 24 , 2006 , the World Health Organization announced the Global Tuberculosis Emergency in 1993 , and noted that effective tuberculosis vaccines should be prioritized .





The only vaccine used to prevent tuberculosis is BCG , which is the attenuated live vaccine strain of Mycobacterium species . Because of the repeated passage , some important immune protective antigen genes of BCG have been mutated , and the immune protection effect is too high . The difference of immune protection force among different populations is very large , which is 0 - 80 % ;
The results of BCG and development of new vaccines have become the focus of research . At present , tuberculosis vaccine is mainly composed of recombinant BCG , subunit vaccine , gene vaccine and nutrition deficient vaccine .





Interleukin - 12 ( IL - 12 ) is a cytokine produced by monocytes and macrophages . It has a wide range of biological activities . It is a functional bridge linking innate immunity and acquired immunity . It is the most widely used cytokine in the induction and regulation of immune active cells in vivo .
It plays an important role in the development of Th1 - type cells by inducing Th0 cells , which can obviously enhance cellular immunity .





IL - 12 was cloned into eukaryotic expression plasmid pcDNA3.1 ( + ) / myc - His to obtain IL - 12 p40 , p35 and p40 - p35 fusion gene . The IL - 12 gene was cloned into eukaryotic expression plasmid pcDNA3.1 ( + ) / myc - His .





IL - 12 and ESAT - 6 were immunized with ESAT - 6 gene . The results showed that IL - 12 and ESAT - 6 were immunized with ESAT - 6 gene . The results showed that IL - 12 and ESAT - 6 were immunized with ESAT - 6 gene and stimulated the organism to produce stronger and stable cellular immune response . The dose of ESAT - 6 was half that of ESAT - 6 alone , but the induced immune response was stronger than that of ESAT - 6 alone , but it was comparable to BCG vaccination .





In addition , the recombinant BCG strain rBCG - 12 capable of secreting IL - 12 gene was further screened by the recombinant BCG - induced SDS - PAGE electrophoresis . The recombinant BCG strain rBCG - 12 expressing IL - 12 gene was further screened by recombinant BCG - induced SDS - PAGE electrophoresis .





rBCG - 12 and BCG were subcutaneously injected into BALB / c mice ( 10 - 6 CFU ) . The levels of serum - specific antibody , spleen lymphocyte proliferation , IFN - 緯 and IL - 4 secretion were measured at 6 , 8 , 10 and 12 weeks after immunization respectively .
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王英;結(jié)核桿菌ESAT6基因密碼子優(yōu)化真核載體的構(gòu)建及其免疫效應(yīng)的研究[D];蚌埠醫(yī)學(xué)院;2013年

本文編號：1986846