發(fā)布時(shí)間：2018-05-21 09:36

  本文選題：結(jié)核桿菌 + ESAT-6��； 參考：《南京醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 第一部分結(jié)核桿菌ESAT-6抗原多表位DNA疫苗的構(gòu)建與體外表達(dá)的鑒定 目的:構(gòu)建含3個(gè)結(jié)核桿菌ESAT-6抗原T細(xì)胞表位及Flt3配體基因的重組質(zhì)粒,并使其在大鼠腎小球系膜細(xì)胞(GMCs)中表達(dá)。方法:用計(jì)算機(jī)軟件預(yù)測(cè)結(jié)核桿菌ESAT-6抗原的T細(xì)胞表位譜,選取3個(gè)T細(xì)胞表位,并以Ala-Ala-Tyr(AAY)序列作為接頭,合成全基因序列,定向克隆入真核雙表達(dá)載體pIRES及質(zhì)粒pIRES-FL。在酶切分析與序列測(cè)定后,用PEI轉(zhuǎn)染至GMCs細(xì)胞,并行Western blot鑒定其體外表達(dá)。結(jié)果:核酸序列測(cè)定證實(shí)重組質(zhì)粒構(gòu)建正確,Westernblot證實(shí)該重組質(zhì)粒能在體外GMCs細(xì)胞中表達(dá)融合蛋白。結(jié)論:成功構(gòu)建了結(jié)核桿菌ESAT-6抗原多表位基因重組質(zhì)粒。 第二部分結(jié)核桿菌ESAT-6抗原多表位DNA疫苗誘導(dǎo)C57BL/6小鼠免疫應(yīng)答的初步研究 目的:研究結(jié)核桿菌ESAT-6抗原多表位基因及FL胞外段共表達(dá)質(zhì)粒對(duì)小鼠的免疫功能的影響。方法:將pIRES-TH、pIRES-TH-FL重組質(zhì)粒免疫小鼠,檢測(cè)小鼠體內(nèi)特異性淋巴細(xì)胞增殖、Th1與Th2型細(xì)胞因子(IFN-γ、IL-2、IL-4、IL-10)分泌以及小鼠血清ESAT-6特異性IgG2a、IgG1型抗體的水平。結(jié)果:經(jīng)pIRES-TH質(zhì)粒免疫的小鼠Th1型應(yīng)答水平上調(diào),Th2型應(yīng)答水平下降。聯(lián)合FL基因的pIRES-TH-FL重組DNA疫苗免疫效果高于單純ESAT-6表位的DNA疫苗。結(jié)論:結(jié)核桿菌ESAT-6表位及FL胞外段共表達(dá)質(zhì)粒能夠提高小鼠體內(nèi)的細(xì)胞免疫功能。
[Abstract]:Part I Construction and in Vitro expression of ESAT-6 Antigen Multiepitope DNA Vaccine of Mycobacterium tuberculosis Aim: to construct three recombinant plasmids containing T cell epitopes and Flt3 ligand genes of ESAT-6 antigen of Mycobacterium tuberculosis and express them in rat glomerular Mesangial cells. Methods: the T cell epitopes of ESAT-6 antigen of Mycobacterium tuberculosis were predicted by computer software. Three T cell epitopes were selected and Ala-Tyr-AAY sequence was used as the junction. The whole gene sequence was synthesized and cloned into eukaryotic double expression vector pIRES and plasmid pIRES-FL. After restriction endonuclease analysis and sequencing, PEI was transfected into GMCs cells and its expression was identified by Western blot in vitro. Results: nucleic acid sequencing confirmed that the recombinant plasmid was constructed correctly and Western blot confirmed that the recombinant plasmid could express fusion protein in GMCs cells in vitro. Conclusion: the recombinant plasmid of ESAT-6 antigen polyepitope gene of Mycobacterium tuberculosis was successfully constructed. The second part: a preliminary study on the immune response of C57BL/6 mice induced by multi-epitope DNA vaccine against ESAT-6 antigen of Mycobacterium tuberculosis Aim: to study the effect of polyepitope gene of ESAT-6 antigen of Mycobacterium tuberculosis and co expression plasmid of extracellular segment of FL on immune function of mice. Methods: the recombinant plasmid pIRES-THS-TH-FL was used to immunize mice. The secretion of Th1 and Th2 type cytokines (IFN- 緯, IL-2IL-4, IL-4, IL-10) and the level of serum IgG2a- IgG1 antibody in mice were detected by immunizing mice with the recombinant plasmid pIRES-TH-FL. Results: the Th1 type response level of mice immunized with pIRES-TH plasmid up-regulated the level of Th2 type response. The immune effect of pIRES-TH-FL recombinant DNA vaccine combined with FL gene was higher than that of DNA vaccine with ESAT-6 epitope alone. Conclusion: the co-expression plasmid of ESAT-6 epitope and extracellular segment of FL can improve the cellular immune function in mice.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392

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相關(guān)期刊論文 前2條

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本文編號(hào)：1918665