结核杆菌ESAT-6抗原多表位DNA疫苗的制备及其免疫效果的初步研究
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摘要
第一部分结核杆菌ESAT-6抗原多表位DNA疫苗的构建与体外表达的鉴定
目的:构建含3个结核杆菌ESAT-6抗原T细胞表位及Flt3配体基因的重组质粒,并使其在大鼠肾小球系膜细胞(GMCs)中表达。方法:用计算机软件预测结核杆菌ESAT-6抗原的T细胞表位谱,选取3个T细胞表位,并以Ala-Ala-Tyr(AAY)序列作为接头,合成全基因序列,定向克隆入真核双表达载体pIRES及质粒pIRES-FL。在酶切分析与序列测定后,用PEI转染至GMCs细胞,并行Western blot鉴定其体外表达。结果:核酸序列测定证实重组质粒构建正确,Westernblot证实该重组质粒能在体外GMCs细胞中表达融合蛋白。结论:成功构建了结核杆菌ESAT-6抗原多表位基因重组质粒。
第二部分结核杆菌ESAT-6抗原多表位DNA疫苗诱导C57BL/6小鼠免疫应答的初步研究
目的:研究结核杆菌ESAT-6抗原多表位基因及FL胞外段共表达质粒对小鼠的免疫功能的影响。方法:将pIRES-TH、pIRES-TH-FL重组质粒免疫小鼠,检测小鼠体内特异性淋巴细胞增殖、Th1与Th2型细胞因子(IFN-γ、IL-2、IL-4、IL-10)分泌以及小鼠血清ESAT-6特异性IgG2a、IgG1型抗体的水平。结果:经pIRES-TH质粒免疫的小鼠Th1型应答水平上调,Th2型应答水平下降。联合FL基因的pIRES-TH-FL重组DNA疫苗免疫效果高于单纯ESAT-6表位的DNA疫苗。结论:结核杆菌ESAT-6表位及FL胞外段共表达质粒能够提高小鼠体内的细胞免疫功能。
PARTⅠConstruction and expression of multiepitope DNA vaccine on ESAT-6 antigen of Mycobacterium tuberculosis in vitro
Objective: To construct recombinant plasmids containing 3 T cell epitopes on ESAT-6 antigen of Mycobacterium tuberculosis and extra-cellular fragment of Flt3 ligand (FL) genes, and to express them in rat glomerular mesangial cells (GMCs). Methods: The amino acid sequence of ESAT-6 antigen was analysed using predictive algorithms and 3 T cell epitopes were predicted. The oligonucleotide encoding and the linker Ala-Ala-Tyr (AAY) were synthesized and inserted into the bicistronic vector pIRES and pIRES-FL. The recombinant plasmids were transfected into GMCs, the recombinant proteins expressing in GMCs was examined by Western blot. Results: The recombinant plasmids were verified by sequencing and the recombinant fusion proteins were identified by Western blot. Conclusion: The multiepitope DNA vaccine from ESAT-6 antigen of Mycobacterium tuberculosis was constructed and expressed successfully.
PARTⅡImmunol effects of gene vaccine constituted with epitopes of tuberculosis ESAT-6 antigen and Flt-3 ligand in mice
Objective: To study the immunol effects of gene vaccine constituted with epitopes of tuberculosis ESAT-6 antigen and Flt-3 ligand in mice. Methods: C57BL/6 mice were inoculated with 100μg of pIRES-TH and pIRES-TH-FL each time and a total of 3 times as a whole. The spleen cell proliferation responses to antigen, the Th1/Th2 responses and the titer of ESAT-6 specific IgG2a/IgGl antibody were examined. Results: The spleen cell proliferation responses, the Th1 responses and the antibody level of ESAT-6 specific IgG2a/IgG1 of the pIRES-TH-FL group are higher than those of control groups. Conclusion: pIRES-TH-FL can effectively elevate the cellular immunity of C57BL/6 mice.
目的:构建含3个结核杆菌ESAT-6抗原T细胞表位及Flt3配体基因的重组质粒,并使其在大鼠肾小球系膜细胞(GMCs)中表达。方法:用计算机软件预测结核杆菌ESAT-6抗原的T细胞表位谱,选取3个T细胞表位,并以Ala-Ala-Tyr(AAY)序列作为接头,合成全基因序列,定向克隆入真核双表达载体pIRES及质粒pIRES-FL。在酶切分析与序列测定后,用PEI转染至GMCs细胞,并行Western blot鉴定其体外表达。结果:核酸序列测定证实重组质粒构建正确,Westernblot证实该重组质粒能在体外GMCs细胞中表达融合蛋白。结论:成功构建了结核杆菌ESAT-6抗原多表位基因重组质粒。
第二部分结核杆菌ESAT-6抗原多表位DNA疫苗诱导C57BL/6小鼠免疫应答的初步研究
目的:研究结核杆菌ESAT-6抗原多表位基因及FL胞外段共表达质粒对小鼠的免疫功能的影响。方法:将pIRES-TH、pIRES-TH-FL重组质粒免疫小鼠,检测小鼠体内特异性淋巴细胞增殖、Th1与Th2型细胞因子(IFN-γ、IL-2、IL-4、IL-10)分泌以及小鼠血清ESAT-6特异性IgG2a、IgG1型抗体的水平。结果:经pIRES-TH质粒免疫的小鼠Th1型应答水平上调,Th2型应答水平下降。联合FL基因的pIRES-TH-FL重组DNA疫苗免疫效果高于单纯ESAT-6表位的DNA疫苗。结论:结核杆菌ESAT-6表位及FL胞外段共表达质粒能够提高小鼠体内的细胞免疫功能。
PARTⅠConstruction and expression of multiepitope DNA vaccine on ESAT-6 antigen of Mycobacterium tuberculosis in vitro
Objective: To construct recombinant plasmids containing 3 T cell epitopes on ESAT-6 antigen of Mycobacterium tuberculosis and extra-cellular fragment of Flt3 ligand (FL) genes, and to express them in rat glomerular mesangial cells (GMCs). Methods: The amino acid sequence of ESAT-6 antigen was analysed using predictive algorithms and 3 T cell epitopes were predicted. The oligonucleotide encoding and the linker Ala-Ala-Tyr (AAY) were synthesized and inserted into the bicistronic vector pIRES and pIRES-FL. The recombinant plasmids were transfected into GMCs, the recombinant proteins expressing in GMCs was examined by Western blot. Results: The recombinant plasmids were verified by sequencing and the recombinant fusion proteins were identified by Western blot. Conclusion: The multiepitope DNA vaccine from ESAT-6 antigen of Mycobacterium tuberculosis was constructed and expressed successfully.
PARTⅡImmunol effects of gene vaccine constituted with epitopes of tuberculosis ESAT-6 antigen and Flt-3 ligand in mice
Objective: To study the immunol effects of gene vaccine constituted with epitopes of tuberculosis ESAT-6 antigen and Flt-3 ligand in mice. Methods: C57BL/6 mice were inoculated with 100μg of pIRES-TH and pIRES-TH-FL each time and a total of 3 times as a whole. The spleen cell proliferation responses to antigen, the Th1/Th2 responses and the titer of ESAT-6 specific IgG2a/IgGl antibody were examined. Results: The spleen cell proliferation responses, the Th1 responses and the antibody level of ESAT-6 specific IgG2a/IgG1 of the pIRES-TH-FL group are higher than those of control groups. Conclusion: pIRES-TH-FL can effectively elevate the cellular immunity of C57BL/6 mice.
引文
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2. Brewer, TF. Preventing tuberculosis with bacillus Calmette-Guerin vaccine: a meta-analysis of the literature [J]. Clin Infect Dis, 2000; 31 (suppl 3): S64-S67.
3. Andersen P, Doherty TM. The success and failure of BCG implications for a novel tuberculosis vaccine [J]. Nat Rev Microbiol, 2005; 3 (8): 656-662.
4. Sorensen AL, Nagai S, Hoven G, et al. Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis [J]. Infect Immun, 1995; 63 (5): 1710-1717.
5. Wang QM, Sun SH, Hu ZL, et al. Improved immunogenicity of a tuberculosis DNA vaccine encoding ESAT6 by DNA priming and protein boosting [J]. Vaccine, 2004; 22 (27-28): 3622- 3627.
6. Disis ML, Rinn K, Knutson KL, Davis D, Caron D, Dela Rosa C, Schiffman K. Flt3 ligand as a vaccine adjuvant in association with HER-2/neu peptide-based vaccines in patients with HER-2/neu-overexpressing cancers [J]. Blood, 2002; 99 (8): 2845-2850.
7. Chen W, Chan AS, Dawson AJ, et al. FLT3 ligand administration after hematopoietic cell transplantation increases circulating dendritic cell precursors that can be activated by CpG oligodeoxy- nucleotides to enhance T-cell and natural killer cell function [J]. Biol Blood Marrow Transplant, 2005; 11(1): 23-34.
8. Sang H, Pisarev VM, Munger C, et al. Regional, but not systemic recruitment/ expansion of dendritic cells by a pluronic-formulated Flt3-ligand plasmid with vaccine adjuvant activity [J]. Vaccine, 2003; 21 (21-22): 3019-3029.
9. Wang A, Braun SE, Soupavde G, Cornetta K. Antileukemic activity of Flt3 ligand in murine leukemia [J]. Cancer Res, 2000; 60 (7): 1895-1900.
10. Sailaja G, Husain S, Nayak BP, Jabbar AM. Long-term maintenance of gpl20-specific immune responses by genetic vaccination with the HIV-1 envelope genes linked to the gene encoding Flt-3 ligand [J]. J Immunol, 2003; 170 (5): 2496-2507.
11. Ling L, Sette A. The multivalent minigene approach to vaccine development [J]. Expert Opin Investig Drugs, 1999; 8 (9): 1351-1357.
12. Wilson CC, McKinney D, Anders M, et al. Development of a DNA vaccine designed to induce cytotoxic T lymphocyte responses to multiple conserved epitopes in HIV-1 [J]. J Immunol, 2003; 171 (10): 5611-5623.
1.Corbett EL,Watt CJ,Walker N,et al.The growing burden of tuberculosis:global trends and interactions with the HIV epidemic [J].Arch Intern Med,2003;163(9):1009-1021.
2.Hesseling,AC,Marais,BJ,Gie,RP,et al.The risk of disseminated Bacille Calmette-Guerin(BCG)disease in HIV-infected children[J].Vaccine,2007;25(1):14-18.
3.Brewer,TF.Preventing tuberculosis with bacillus Calmette-Guerin vaccine:a meta-analysis of the literature[J].Clin Infect Dis,2000;31(suppl 3):S64-S67.
4.Andersen P,Doherty TM.The success and failure of BCG implications for a novel tuberculosis vaccine[J].Nat Rev Microbiol,2005;3(8):656-662.
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