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

【摘要】： 第一部分結核桿菌ESAT-6抗原多表位DNA疫苗的構建與體外表達的鑒定 目的:構建含3個結核桿菌ESAT-6抗原T細胞表位及Flt3配體基因的重組質粒,并使其在大鼠腎小球系膜細胞(GMCs)中表達。方法:用計算機軟件預測結核桿菌ESAT-6抗原的T細胞表位譜,選取3個T細胞表位,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載體pIRES及質粒pIRES-FL。在酶切分析與序列測定后,用PEI轉染至GMCs細胞,并行Western blot鑒定其體外表達。結果:核酸序列測定證實重組質粒構建正確,Westernblot證實該重組質粒能在體外GMCs細胞中表達融合蛋白。結論:成功構建了結核桿菌ESAT-6抗原多表位基因重組質粒。 第二部分結核桿菌ESAT-6抗原多表位DNA疫苗誘導C57BL/6小鼠免疫應答的初步研究 目的:研究結核桿菌ESAT-6抗原多表位基因及FL胞外段共表達質粒對小鼠的免疫功能的影響。方法:將pIRES-TH、pIRES-TH-FL重組質粒免疫小鼠,檢測小鼠體內特異性淋巴細胞增殖、Th1與Th2型細胞因子(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。結果:經(jīng)pIRES-TH質粒免疫的小鼠Th1型應答水平上調,Th2型應答水平下降。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。結論:結核桿菌ESAT-6表位及FL胞外段共表達質粒能夠提高小鼠體內的細胞免疫功能。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

【參考文獻】

相關期刊論文 前2條

1 楊永林,張學光,黃祖瑚;乙型肝炎病毒核心抗原及Flt3配體雙表達核酸疫苗的構建與體外表達[J];南京醫(yī)科大學學報(自然科學版);2005年05期

2 徐聞歡;徐娟;王迎偉;陳霞;高玲娟;吳宜琴;;結核桿菌ESAT-6抗原及Flt3配體雙表達核酸疫苗的構建與體外表達[J];南京醫(yī)科大學學報(自然科學版);2007年01期

，

本文編號：1918665