结核分支杆菌HSP65的基因表达及其免疫学特性研究
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摘要
结核分支杆菌(Mycobacterium tuberculosis, MTB)热休克蛋白6(5Heat Shock Protein 65, HSP65)是热休克蛋白60家族成员之一,由groEL2基因编码,是一种高度保守的蛋白质。该蛋白具有很强的免疫原性,在结核杆菌感染过程中,HSP65是机体对抗其入侵的重要的免疫保护性抗原之一,提示HSP65可能成为结核病预防和治疗的一个关键因素。本研究采用分子生物学方法分别构建MTB HSP65的原核重组质粒和真核重组质粒,测定分析了HSP65蛋白和真核重组质粒在小鼠体内的免疫学特性和抵抗MTB感染的保护力。
1.采用PCR方法从MTB H37Rv株DNA基因组中扩增HSP65基因,与pMD18-T载体连接,酶切鉴定阳性重组质粒pMD-hsp65,经测序证实与Genebank公布的MTB HSP65基因序列完全一致。利用酶切位点将HSP65基因从pMD-hsp65中亚克隆入pPRO EX HTa原核表达载体,酶切鉴定阳性重组质粒pPRO-hsp65,并用IPTG进行诱导表达。对表达产物进行SDS-PAGE的结果表明,在相对分子量为65 Ku的位置有目的蛋白表达。用抗MTB HSP65的mAb及TB患者血清进行Western-blot鉴定,结果证实在相应的位置有特异性的结合条带。采用Ni-NTA亲和色谱柱在变性条件下纯化得到了HSP65蛋白。
2.利用酶切位点将HSP65基因从pMD18-hsp65中亚克隆入真核表达载体pcDNA3.1(-),酶切鉴定阳性重组质粒pcDNA-hsp65。将重组质粒用阳离子聚合物法稳定转染P815(H-2d)细胞。分别经RT-PCR和间接免疫荧光法证实,该重组质粒可在稳定转染的P815细胞中转录和特异性地表达。经过G418压力筛选,得到8株能稳定表达HSP65蛋白的P815细胞系。
3.用HSP65原核表达蛋白及真核重组质粒免疫BALB/c小鼠,并检测两种疫苗各项免疫学指标。结果表明,HSP65亚单位疫苗和基因疫苗均可在小鼠体内刺激产生较强的体液和细胞免疫应答,并各有特点;均可显著减少感染小鼠体内的脾荷菌数,表明其可抵抗MTB的感染,但这种保护作用均未超过BCG。
上述结论为进一步研究HSP65疫苗的免疫学特性及与其他疫苗的优势组合,用于TB新型疫苗的开发提供了实验数据和物质基础。
Mycobacterium tuberculosis (MTB) Heat Shock Protein 65(HSP65)which belongs to HSP60s,encoding by gene groEL2 ,is a kind of highly conservative protein. It is one of the important protective antigens for resistance of MTB infection. The protein has strong immunogenicity which has been proved that about 20% reactive T cell can discriminate HSP65 in MTB infected mouse, thus HSP65 may a key point for precaution and therapy of tuberculosis (TB).
This research used the molecular biology method to construct prokaryotic recombinant plasmid and eukaryotic recombinant plasmid based on HSP65 of MTB .Compared with BCG, to evaluate the immunological properties and protective efficacy of them against MTB infection, in order to find a new efficient vaccine against TB. The results as follows:
1. The gene of HSP65 was amplified from DNA genome of Mycobacterium tuberculosis H37Rv strain by polymerase chain reaction (PCR). Inserted the PCR product into pMD18-T vector and sequenced. Ligated the right sequenced HSP65 gene into pPRO EX HTa prokaryotic expression vector and identified by restrictive enzyme digestion, the recombinant plasmid was named pPRO-hsp65. The recombinant plasmid was transformed into E.coli DH5αstrain and induced with IPTG.The analysis of SDS-PAGE showed that there was a specific protein expression at 65Ku molecular marker, and the protein was further identified by Western-blot using anti- HSP65 mAb and serum of TB patient. Purified the fusion protein by Ni-NTA purification system.
2. Subcloned the gene of HSP65 into eukaryotic expression vector pcDNA3.1 (-), the recombinant plasmid was named pcDNA-hsp65 and identified by restrictive enzyme digestion.Transfected the recombinant plasmid into P815 cells stably by Suohua-sofast transfection reagent. The specific mRNA of the gene was detected by RT-PCR, and the expression of HSP65 protein in the transfected cells was detected by indirect immunofluorescence technique (IIFT). The result showed that there were specific strap in the agarose gel electrophoresis (AGE) and green immunofluorescence showed in the transfected P815 cells. Eight lines of transfected P815 cells stably expressing the protein of HSP65 was selected by G418.
3. Immunized BALB / c mice with prokaryotic expression protein and eukaryotic recombinant plasmid of HSP65 gene, and tested the two vaccines immunological parameters. Results showed that, both the subunit and DNA vaccine of HSP65 protein could elicite great humoral and cellular immune responses, and had their own characteristics. The two vaccines could significantly reduce the bacteria loads in spleen and lungs of infected mice, this meant that the vaccines could prevent the mice against MTB infection, but the ability of protection was not better than that of BCG.
All these conclusions brought some new clues for researching prevention and treatment of tuberculosis.
1.采用PCR方法从MTB H37Rv株DNA基因组中扩增HSP65基因,与pMD18-T载体连接,酶切鉴定阳性重组质粒pMD-hsp65,经测序证实与Genebank公布的MTB HSP65基因序列完全一致。利用酶切位点将HSP65基因从pMD-hsp65中亚克隆入pPRO EX HTa原核表达载体,酶切鉴定阳性重组质粒pPRO-hsp65,并用IPTG进行诱导表达。对表达产物进行SDS-PAGE的结果表明,在相对分子量为65 Ku的位置有目的蛋白表达。用抗MTB HSP65的mAb及TB患者血清进行Western-blot鉴定,结果证实在相应的位置有特异性的结合条带。采用Ni-NTA亲和色谱柱在变性条件下纯化得到了HSP65蛋白。
2.利用酶切位点将HSP65基因从pMD18-hsp65中亚克隆入真核表达载体pcDNA3.1(-),酶切鉴定阳性重组质粒pcDNA-hsp65。将重组质粒用阳离子聚合物法稳定转染P815(H-2d)细胞。分别经RT-PCR和间接免疫荧光法证实,该重组质粒可在稳定转染的P815细胞中转录和特异性地表达。经过G418压力筛选,得到8株能稳定表达HSP65蛋白的P815细胞系。
3.用HSP65原核表达蛋白及真核重组质粒免疫BALB/c小鼠,并检测两种疫苗各项免疫学指标。结果表明,HSP65亚单位疫苗和基因疫苗均可在小鼠体内刺激产生较强的体液和细胞免疫应答,并各有特点;均可显著减少感染小鼠体内的脾荷菌数,表明其可抵抗MTB的感染,但这种保护作用均未超过BCG。
上述结论为进一步研究HSP65疫苗的免疫学特性及与其他疫苗的优势组合,用于TB新型疫苗的开发提供了实验数据和物质基础。
Mycobacterium tuberculosis (MTB) Heat Shock Protein 65(HSP65)which belongs to HSP60s,encoding by gene groEL2 ,is a kind of highly conservative protein. It is one of the important protective antigens for resistance of MTB infection. The protein has strong immunogenicity which has been proved that about 20% reactive T cell can discriminate HSP65 in MTB infected mouse, thus HSP65 may a key point for precaution and therapy of tuberculosis (TB).
This research used the molecular biology method to construct prokaryotic recombinant plasmid and eukaryotic recombinant plasmid based on HSP65 of MTB .Compared with BCG, to evaluate the immunological properties and protective efficacy of them against MTB infection, in order to find a new efficient vaccine against TB. The results as follows:
1. The gene of HSP65 was amplified from DNA genome of Mycobacterium tuberculosis H37Rv strain by polymerase chain reaction (PCR). Inserted the PCR product into pMD18-T vector and sequenced. Ligated the right sequenced HSP65 gene into pPRO EX HTa prokaryotic expression vector and identified by restrictive enzyme digestion, the recombinant plasmid was named pPRO-hsp65. The recombinant plasmid was transformed into E.coli DH5αstrain and induced with IPTG.The analysis of SDS-PAGE showed that there was a specific protein expression at 65Ku molecular marker, and the protein was further identified by Western-blot using anti- HSP65 mAb and serum of TB patient. Purified the fusion protein by Ni-NTA purification system.
2. Subcloned the gene of HSP65 into eukaryotic expression vector pcDNA3.1 (-), the recombinant plasmid was named pcDNA-hsp65 and identified by restrictive enzyme digestion.Transfected the recombinant plasmid into P815 cells stably by Suohua-sofast transfection reagent. The specific mRNA of the gene was detected by RT-PCR, and the expression of HSP65 protein in the transfected cells was detected by indirect immunofluorescence technique (IIFT). The result showed that there were specific strap in the agarose gel electrophoresis (AGE) and green immunofluorescence showed in the transfected P815 cells. Eight lines of transfected P815 cells stably expressing the protein of HSP65 was selected by G418.
3. Immunized BALB / c mice with prokaryotic expression protein and eukaryotic recombinant plasmid of HSP65 gene, and tested the two vaccines immunological parameters. Results showed that, both the subunit and DNA vaccine of HSP65 protein could elicite great humoral and cellular immune responses, and had their own characteristics. The two vaccines could significantly reduce the bacteria loads in spleen and lungs of infected mice, this meant that the vaccines could prevent the mice against MTB infection, but the ability of protection was not better than that of BCG.
All these conclusions brought some new clues for researching prevention and treatment of tuberculosis.
引文
[1] WHO report 2007. Global tuberculosis control: surveillance, planning, financing. World Health Organization.
[2] 刘剑君,幺鸿雁.我国结核病的流行现状和防治对策[J].预防医学论坛,2006,12(5):638-640.
[3] Flynn JL, Chan J. Tuberculosis: latency and reactivation[J]. Infect Immun,2001, 69(7):4195-4201.
[4] Gatfield J, Pieters J. Essential role for cholesterol in entry of Mycobacteria into macrophages [J].Science,2000, 288(5471):1647-1650.
[5] Neyrolles O, Gould K, Gares MP, et al. Lipoprotein access to MHC Class I presentation during infection of routine macrophages with live Mycobacteria[J].Immun, 2001,166(1): 447-457.
[6] Pasula R, Wright JR, Kachel DL, et al. Surfactant protein A suppresses reactive nitrogen intermediates by alveolar macrophages in response to Mycobacterium tuberculosis[J].Clin Invest, 1999,103(4):483-490.
[7] Mahenthiralingam E, Marklund BI, Brooks LA, et al. Site-Directed Mutagenesis of the 19-Kilodalton Lipoprotein Antigen Reveals No Essential Role for the Protein in the Growth and Virulence of Mycobacterium intracellulare[J]. Infect Immun, 1998, 66(8):3626- 3634.
[8] Pieters J. Entry and survival of pathogenic mycobacteria in macrophages[J]. Microbes Infect, 2001,3(3):249-255.
[9] Anand PK, Kaul D. Downregulation of TACO gene transcription restricts mycobacterial entry/survival within human macrophages[J]. FEMS Microbiol Lett, 2005, 250(1):137-144.
[10] Voss JJD, Rutter K, Schroeder BG, et al. Iron Acquisition and Metabolism by Mycobacteria[J]. Bacteriol, 1999,181(15):4443-4451.
[11] Perez E, Samper S, Bordas Y, et al. An essential role for phoP in Mycobacterium tuberculosis virulence[J]. Mol Microbiol, 2001, 41(1):179-187.
[12] Braunstein M, Espinosa BJ, Chan J, et al. SecA2 functions in the secretion of superoxide dismutase A and in the virulence of Mycobacterium tuberculosis[J]. Mol Microbiol, 2003, 48(2): 453-464.
[13] Boom WH, Chervenal KA, Mincek MA, et al. Role of the mononuclear phagocyte as an antigen-presenting cell for human cells activated by live mycobacterium tuberculosis[J].Infect Immun,1992,60(9):3480-3488.
[14] Steffen Stenger, Modlin RL. T cell mediated immunity to Mycobacterium tuberculosis[J].Curr Opin in Microbiol,1999, 2(1):89-93.
[15] Flynn JL, Ernst JD. Immune responses in tuberculosis[J]. Curr Opin Immunol, 2000,12(4):432-436.
[16] Kurts C, Carbone FR, Barnden M, et al. CD4+T Cell Help Impairs CD8+T Cell Deletion Induced by Cross-presentation of Self-Antigens and Favors Autoimmunity[J]. Exp Med, 1997, 186 (12):2057-2062.
[17] Lazarevic V, Flynn J. CD8+T Cells in Tuberculosis[J]. Am J Respir Crit Care Med, 2002,166(8):1116-1121.
[18] Havlir DV, Ellner JJ, Chervenak KA, et al. Selective expansion of human gamma delta T cells bynon-peptide antigens and Daudi cells analyzed by TCR gene transfer[J]. Immunol,1995,154(3):998-1006.
[20] ohnson BJ, Ress SR, Willcox P, et al. Clinical and immune responses of tuberculosis patients treated with low-dose IL-2 and multidrug therapy[J]. Cytokines Mol Ther, 1995,1(3):185-196.
[21] Shingo H, Naoyuki N, Takeshi K, et al. Preliminary evaluation of soluble IL-2 receptor and type III procollagen N-terminal aminopeptide in pleural fluid for differentiating tuberculous, carcinomatous and parapneumonic pleural effusions[J]. Respirology, 2002,7(4):311-315.
[22] Gazzinell RT, Wysocka M, Hayashi S, et al. Parasite-induced IL-12 stimulates early synthesis and resistance during acute infection with Toxoplasma gondii[J]. Immunol, 1994, 153(6):2533-2543.
[23] Rhoades ER, Goopper AM, Omne IM. Chemokine response in mice infected with Mycobacterium tuberculosis[J].Infect Immun,1995,63(10):3871-3877.
[24] Cooper AM, Magram J, Ferrante J, et al. Interleukin-12 (IL-12) is crucial to the development of protective immunity in mice intravenously infected with Mycobacterium tuberculosis[J]. Exp Med,1997, 186(1):39-45.
[25] Flynn JL, Goldstein MM, Triebold KJ, et al. IL-12 increases resistance of BALB/c mice to Mycobacterium tuberculosis infection[J]. Immunol,1995,155(5):2515-2524.
[26] Das G, Sheridan S, Janeway CA. The Source of Early IFN-γ That Plays a Role in Th1 Priming[J]. Immunol, 2001,167(4): 2004-2010.
[27] Lyadova I, Yeremeev V, Majorov K, et al. An ex vivo study of T Lymphocytes recovered from the lungs of I/St mice infected with and susceptible to Mycobacterium tuberculosis[J]. Infec.Immun, 1998,66(10): 4981-4988.
[28] Orme I, Miller E, Roberts A, et al. T Lymphocytes mediating protection and cellular cytolysis during the course of Mycobacterium tuberculosis infection[J]. Immunol,1992,148(1):189-196.
[29] Cooper AM, Dalton DK, Stewart TA, et al. Disseminated tuberculosis in IFN-γ gene-disrupted mice[J]. Exp Med, 178(6): 2243-2248.
[30] Flynn JL, Chan J, Triebold KJ, et al. An essential role for Interferon-γ in resistance to Mycobacterium tuberculosis infection[J]. Exp Med.178(6):2249-2254.
[31] Lalvani A, Brookes R, Wilkinson RJ, et al. Human cytolytic and interferon gamma- secreting CD8 T lymphocytes specific for Mycobacterium tuberculosis[J]. PNAS, 1998, 95(1):270-275.
[32] Ottenhoff TH, Kumararatne D, Casanova JL. Noval human immunodeficiencies reveal the essential role of type-1 cytokines in immunity to intracellular bacteria[J]. Immunol Today, 1998,19(11):491-494.
[33] Ladel CH, Szalay G, Reidel D, et al. Interleukin-12 secretion by Mycobacterium tuberculosis infected macrophages[J].Infect Immun,1997,65(5): 1936-1938.
[34] Henderson RA, Watkins SC, Flynn JL. Activation of human dendritic cells following infection with Mycobacterium tuberculosis[J]. Immunol,1997,159 (2):635-643.
[35] Flynn JL, Goldstein MM, Chan J, et al. Tumor necrosis factor is required in the protective immune response against M.tuberculosis in mice[J].Immunity,1995, 2(6):561-572.
[36] Bean AGD, Roach DR, Briscoe H, et al. Structural deficiencies in granuloma formation in TNF gene targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection ,which is not compensated for by lymphotoxin[J]. Immunol,1999,162(6):3504-3511.
[37] Moreira AL, Tsenova L, Wang J, et al. Effect of cytokine modulation by thalidomide on the gramomatous response in murine tuberculosis[J].Tubercle Lung Disease, 1997,78(1):47-55.
[38] Hodge DJ, Marino MW, Horton MR, et al. Inhibition of interferon induced interleukin 12 production:a potential mechanism for the anti-inflammatory activities of tumor necrosis factor[J].PNAS, 1998,95(23): 13806-13811.
[39] Fenhalls G, Wong A, Bezuidenhout J, et al. In situ production of gamma interferon,interleukin-4,and tumor necrosis factor alpha mRNA in human lung tuberculous granuloma[J].Infect Immun, 2000,68(5):2827- 2836.
[40] Gong J, Zhang M, Modlin R, et al. Interleukin-10 downregulates Mycobacterium tuberculosis-induced Th1 responses and CTLA4 expression[J]. Infect Immun, 1996,64(3):913-918.
[41] Rojas M, Olivier M, Gros P, et al. TNFα and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages[J].Immunol, 1999,162(10):6122-6131.
[42] Ladel R, Miyajima A, Mee PJ, et al. Lethal tuberculosis in interleukin-6-deficient mutant mice[J].Blood,1996,88(7):2458-2464.
[43] Reed JA, Ikegami M, Robb L, et al. Cytokine gene expression by cultures of human lymphocytes with autologous Mycobacterium tuberculosis-infected monocytes[J].AmJ Physiol Lung Cell Mol Physiol,2000,278(6):1164-1171.
[44] Johnson BJ, Ress SR, Willcox P, et al. Clinical and immune responses of tuberculosis patients treated with low-dose IL-2 and multidrug therapy[J]. Cytokines Mol Ther, 1995,1(3):185-196.
[45] Fine PE. Variation in protection by BCG: implications of and for heterologous immunity [J].Lancet,1995,346(8986):1339-1345.
[46] Rook GA, Dheda K, ZumLa A. Immune responses to tuberculosis in developing countries: implications for new vaccines[J]. Nat Rev Immunol 2005,5(8):661-667.
[47] Brooks JV, Frank AA, Keen MA, et al. Boosting vaccine for tuberculosis[J]. Infect Immu -n,2001,69(4):2714-2717.
[48] van Rie A, Warren R, Richardson M, et al. Exogenous Reinfection as a Cause of Recurrent Tuberculosis after Curative Treatment[J]. N Engl J Med,1999,341(16):1174- 1179.
[49] Behr MA, Wilon MA, Gill WP, et al. Comparative genomics of BCG vaccines By whole genome DNA microarry[J].Science,1999,284(5419):1520-1523.
[50] Lewis KN, Liao R, Guinn KM, et al. Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guerin attenuation[J]. Infect Dis, 2003, 187(1):117-123.
[51] Orme IM, Collins FM. Efficacy of Mycobacterium bovis BCG vaccination in mice undergoing prior pulmonary infection with atypical mycobacteria. Infect[J]. Immun, 1984,44(1):28-32.
[52] Hubbard RD, Flory CM, FM Collins. Immunization of mice with mycobacterial culture filtrate proteins[J].Clin Exp Immunol, 1992,7(1):94-98.
[53] Sonnenberg MG, Belisle JT. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N- terminal amino acid sequencing, and electrospray mass spectr- ometry[J].Infect Immun,1997,65(1):4515-4524.
[54] Andersen P, Andersen AB, Sorensen AL, et al. Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice[J]. Immunol, 1995,154(7):3359-3372.
[55] Li ZM, Howard A, Kelley C, et al. Immunogenicity of DNA vaccines expressing tuberculosis proteinsfused to tissue plasminogen activator signal sequences[J]. Infect Immun,1999,67(9):4780-4786.
[56] Zhu XJ, Venkataprasad N, Thangaraj HS, et al. Functions and specificity of T ells following nucleic acid vaccination of mice against Mycobacterium tuberculosis[J]. Immunol,1997, 158(12):5921-5926.
[57] Erb KJ, Kirman J, Woodfield L, et al. Identification of potential CD8+T-cell epitopes of the 19kua and AhpC proteins from Mycobacterium tuberculosis. No evidence for CD8+ T-cell priming against the identified peptides after DNA-vaccination of mice[J]. Vaccine,1998,16(7):692-697.
[58] Anderson P, Askgaard D, Gottschau A, et al. Identification of immunodominant antigens during infection with Mycobacterium tuberculosis[J]. Scand J Immunol,1992,36(6):823-831.
[59] Olsen AW, Laurens AH, Pinxteren V, et al. Protection of Mice with a Tuberculosis Subunit Vaccine Based on a Fusion Protein of Antigen 85B and ESAT-6[J]. Infect Immun,2001,69(5):2773-2778.
[60] Horwitz MA, Lee BW, Dillon BJ, et al. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis[J]. PNAS,1995,92(5):1530-1534.
[61] Lindblad EB, Elhay MJ, Silva R, et al. Adjuvant modulation of immune responses to tuberculosis subunit vaccines.Infect[J]. Immun, 1997,65(2): 623-629.
[62] Sinha RK, Khuller GK. The protective efficacy of a liposomal encapsulated 30 kua secretory protein of Mycobacterium tuberculosis H37Ra against tuberculosis in mice[J].Immunol Cell Biol,1997,75(5): 461-466.
[63] Vordermeier HM, Coombes AG, Jenkins P, et al. Synthetic delivery system for tuberculosis vaccines: immunological evaluation of the M. tuberculosis 38 kua protein entrapped in biodegradable PLG microparticles[J].Vaccine,1995,13(16):1576-582.
[64] Stenger S. An antimicrobial activity of cytolytic T cells mediated by granulysin[J]. Science,1998,282(5386):121-125.
[65] Lowrie DB, Silva CL. Enhancement of immunocompetence in tuberculosis by DNA vaccination [J].Vaccine, 2000(18):1712-1716.
[66] 范雄林, 李元, 徐志凯. 结核分支杆菌基因疫苗研究进展[J].细胞与分子免疫学杂志, 2001,17(4):121-123.
[67] Morris S, Kelley C, Howard A, et al. The immunogenicity of single and combination DNA vaccines against tuberculosis[J].Vaccine,2000,18(20): 2155-2163.
[68] Kamath AT, Feng CG, Macdonald M, et al. Differential Protective Efficacy of DNA Vaccines Expressing Secreted Proteins of Mycobacterium tuberculosis[J]. Infect Immun, 1999, 67(4):1702-1707.
[69] 师长宏, 范雄林, 徐志凯, 等.结核分支杆菌分泌蛋白 Ag85B-ESAT-6 的融合表达及纯化[J].中华结核和呼吸杂志,2004,(2): 89-92.
[70] 师长宏, 范雄林, 徐志凯, 等. 融合表达 Ag85B-ESAT-6 的结核分支杆菌真核表达载体的构建及其免疫原性[J].第四军医大学学报,2004,25(2):121-124.
[71] Bonato VL, Lima VM, Tascon RE, et al. Identification and characterization of protective T cells in hsp65 DNA-vaccined and M. tuberculosis infected mice[J]. Infect Immun, 1998,66(5):169-175.
[72] Tacson RE, Colston MJ, Ragno S, et al. Vaccination against tuberculosis by DNA injection[J].Nat Med,1996,2(8):888- 892.
[73] Kumar V, Sercarz E. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine[J]. NatMed,1996,2(8):857-859.
[74] Ulmer JB, Liu MA, Montgomery DL, et al. Expression and immunogenicity of M.tuberculosis antigen 85 by DNA vaccination[J]. Vaccine,1997,15(8):792-794.
[75] Lozes E, Huygen K, Content J, et al. Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex[J].Vaccine,1997,15(8):830-833.
[76] Denis O, Tanghe A, Palfliet K, et al. Vaccination with plasmid DNA encoding mycobacterial antigen 85A stimulates a CD4+ and CD8+ T-cell epitopic repertoire broader than that stimulated by M. tuberculosis H37Rv infection[J]. Infect Immun, 1998,66(4):1527-1533.
[77] Baldwin SL, D’SOUZA CD, Orme IM, et al. Immunogenicity and protective efficacy of DNA vaccines encoding secreted and non-secreted forms of Mycobacterium tuberculosis Ag85A[J]. Tuber Lung Dis,1999,79(4):251-259.
[78] Dillon DC, Alderson MR, Day CH, et al. Molecular Characterization and human T- cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family[J]. Infect Immun,1999,67(4):2941-2950.
[79] Lefevre P, Denis O, De Wit L, et al. Cloning of the genge encoding a 22-kilodalton cell surface antigen of Mycobacterium bovis BCG and analysis of its potential for DNA vaccination against tuberculosis[J]. Infect Immun, 2000,68(3):1040-1047.
[80] Tanghe A, Lefevre P, Denis O, et al. Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors[J]. Immunol,1999,162(2):1113-1119.
[81] Chambers MA, Vordermeier H, Whelan A, et al. Vaccination of mice and cattle with plasmid DNA encoding the Mycobacterium bovis antigen MPB83[J].Clin Infect Dis,2000,30(Suppl3): S283-287.
[82] Grover A, Ahmed MF, Verma I, et al. Expression and purification of the Mycobacterium tuberculosis complex-restricted antigen CFP21 to study its immunoprophylactic potential in mouse model[J]. Protein Expr Purif, 2006, 48(2): 274-280.
[83] Hervas-Stubbs S, Majlessi L, et al. High Frequency of CD4+ T Cells Specific for the TB10.4 Protein Correlates with Protection against Mycobacterium tuberculosis Infection[J]. Infect. Immun., 2006,74(6):3396-3407.
[84] Coler RN, Campos-Neto A, Ovendale P, et al. Vaccination with the T Cell Antigen Mtb 8.4 Protects Against Challenge with Mycobacterium tuberculosis[J]. Immunol, 2001, 166(10):6227-6235.
[85] Ha SJ, Jeon BY, Kim SC, et al. Therapeutic effect of DNA vaccines combined with chemotherapy in a latent infection model after aerosol infection of mice with Mycobacterium tuberculosis[J]. Gene Ther 2003;10(18):1592-1599.
[86] Ha SJ, Jeon BY, Youn JI, et al. Protective effect of DNA vaccine during chemotherapy on reactivation and reinfection of Mycobacterium tuberculosis[J]. Gene Ther 2005,12(7):634–638.
[87] Silva CL, Bonato VLD, Coelho-Castelo AAM, et al. Immunotherapy with plasmid DNA encoding mycobacterial hsp65 in association with chemotherapy is a more rapid and efficient form of treatment for tuberculosis in mice[J]. Gene Ther 2005;12(3):281-287.
[88] Nuermberger E, Tyagi S, Williams KN, et al. Rifapentine, Moxifloxacin, or DNA Vaccine Improves Treatment of Latent Tuberculosis in a Mouse Model[J]. Am. J Respir Crit Care Med, 2005,172(11): 1452-1456.
[89] Zhu X, Venkataprasad N, Ivanyi J, et al.Vaccination with recombinant vaccinia viruses protects miceagainst Mycobacterium tuberculosis infection[J].Immunology,1997,92(1):6-9.
[90] Hess J, Grode L, Hellwig J, et al. Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kua antigen of Mycobacterium bovis BCG[J]. FEMS Immunol Med Microbiol, 2000, 27(4):283-289.
[91] Chan J, Kaufmann S. Immune mechanisms of protection in tuberculosis pathogenesis, protection and control.Ed[A]. American Society of Microbiology[C]. Washington, D.C: BR Bloom, 1994,9389-9415.
[92] Bange FC, Brown AM, Jacobs WR. Leucine auxotrophy restricts growth of mycobacterium bovis BCG in macrophages[J]. Infect Immun,1996,64(5):1794- 1799.
[93] Smith DA, Parish T, Stoker NG, et al. Characterization of auxotrophic mutants of mycobacterium tuberculosis and their potential as vaccine candidates[J]. Infect Immun,2001,69(2):1142-1150.
[94] Jackson M, Phalen SW, Lagranderie M, et al. Persistence and protective efficacy of mycobacterium tuberculosis auxotroph vaccine[J]. Infect Immun, 1999,67(6): 2867-2873.
[95] Horwitz MA, Harth G, Dillon BJ, et al. Recombinant bacillus Calmette-Guerin(BCG) vaccines expressing the Mycobacterium tuberculosis 30-kua major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model[J].PNAS,2000,97(25):13853-13858.
[96] Bao L, Chen W, Zhang H, et al. Virulence, immunogenicity, and protective efficacy of two recombinant Mycobacterium bovis Bacillus Calmette-Guerin strains expressing the antigen ESAT6 from Mycobacterium tuberculosis[J]. Infect Immun,2003,71(4): 1656-1661.
[97] Hess J, Miko D, Catic A, et al. Mycobacterium bovis Bacille Calmette Guerin strains secreting listeriolysin of Listeria monocytogenes[J].PNAS,1998,95(9):5299-5304.
[98] 赵宝华, 张莉, 石振华.基因重组卡介苗的优点及应用[J].生物学通报. 2002,37(1):18-20.
[99] Hart HG, Lee BY, Horwitz MA. High level heterologous expression and secretion in rapidly growing nonpat hogenic Mycobacteria of four major Mycobacterium tuberculosis ext racellular proteins considered to be leading vaccine candidates and drug targets[J]. Infect Immun,1997,65 (6):2321-2328.
[100] Falcone V, Bassey E, Jacobs W Jr, et al. The immunogenicity of recombinant mycobacterium smegmatis bearing BCG genes[J]. Microbiology, 1995,141(pt5):1239-1245.
[101] 胡佳杰, 文学明, 肖爱清, 等. 重组 BCG-hsp70 疫苗有关毒性实验研究[J]. 广西预防医学,2002,8(4):233-235.
[102] 胡佳杰, 文学明, 肖爱清, 等. 重组 M.S-Sj26GST 疫苗有关毒性实验研究[J].华南预防医学,2002,28(5):44-45.
[103] Stenger S, Mazzaccaro RJ, Uyemura K, et al. Differential effects of cytolytic T-cell subsets on intracellular infection[J]. Science,1997,276(5319): 1684-1687.
[104] Andersen P, Askgaard D, Gottschau A. Identification of immunod ominant antigens during infection with Mycobacterium tuberculosis[J].Scand J Immunol, 1992,36(2):823-828.
[105] Kaufman SHE. The cellular immune response to heat shock proteins[J]. Experientia, 199 2,48(3):640-645.
[106] Charo J, Geluk A, Sundback M, et al. The identification of a comLnon pathogen-specfic HLA class I A*0201-restricted cytolytic T cell epitope encoded within the heat shock protein 65[J].Eur J Immunol, 2001,31(12):3602- 3611.
[107] Ragno S, Colston MJ, Lowrie DB, et al. Protection of rats from adjuvant arthritis by immunization with naked DNA encoding for mycobacterial heat shock protein 65[J]. Arthritis Rheum,1997, 40(2):277-283.
[108] [108] Turner OC, Roberts AD, Fran k AA, et al. Lack of protection in mice an d necrotizing bronchointerstitial pneumonia bronchiolitis in sninea pigs immunized with vaccines directed against the HSP65 molecular of Mycobacterium tuberculosis[J]. Infect Immun,2000,68(6):3674-3679.
[109] 刘思国,王春来,彭永刚,等.牛分支杆菌热休克蛋白 65 基因的分子克隆和表达[J]. 中国预防兽医学报,2004,26(5):336-271.
[110] Shinnick TM. The 65-kilodalton antigen of Mycobacterium tuberculosis[J].Bacteriol, 1987, 169 (3):1080-1088.
[111] H.Martin Vordermeier, Douglas B. Lowrie, R. Glyn Hewinson. Improved immunogenicity of DNA vaccination with mycobacterial HSP65 against bovine tuberculosis by protein boosting[J].Veterinary Microbiology,2003, (93) : 349–359.
[112] Yang BF, Zhao HL, Xue C, et al.Recombinant heat shock protein 65 carrying hepatitis B core antigen induces HBcAg-specific CTL response[J]. Vaccine.,2007, 25(22):4478-86.
[113] Lowrie DB, Tascon RE, Bonato VL, et al. Therapy of tuberculosis in mice by DNA vaccination[J]. Nature, 1999, 400(6741): 269-271.
[114] 居巍, 刘君炎, 庄玉辉, 等. 结核杆菌pCHSP65真核表达载体的构建及其DNA免疫实验[J].中国免疫学杂志, 2003,19(1):12-15.
[115] J.萨姆布鲁克, EF.弗里奇, T.曼尼阿蒂斯. 分子克隆实验指南[M]. 北京: 科学出版社,1996.49-55.
[116] Silva C L, Lowrie D B. A single mycobacterial protein (hsp65) expressed by a transgenic antigen-presenting cell vaccinates mice against tuberculosis[J] . Immunology, 1994, 82:244.
[117] Lowrie D B, Silva C L, Colston MJ, et al. Protection against by a plasmid DNA vaccine [J]. Vaccine, 1997, 15:834.
[118] Schirmbeck R, Melber K, Kuhrober A, et al. Immunization with soluble hepatitis B virus surface protein elicits murine H-2 class I-restricted CD8+ cytotoxic T lymphocyte responses in vivo[J]. Immunol, 1994,152(3):1110-1119.
[119] Stewart MJ, Plautz GE, Del Buono L, et al. Gene transfer in vivo with DNA-liposome complexes: safety and acute toxicity in mice[J]. Hum Gene Ther, 1992,3(3):267-275.
[120] Elhay H,Martin J,Ersen et al.Immunological requirements for a subunit vaccine against tuberculosis [J].Immunology and Cell Biology.1997,75(6):595-603.
[121] 薛莹.结核分支杆菌Rpf样蛋白生物学及免疫学活性的研究[D].第四军医大学博士论文.2005年.
[2] 刘剑君,幺鸿雁.我国结核病的流行现状和防治对策[J].预防医学论坛,2006,12(5):638-640.
[3] Flynn JL, Chan J. Tuberculosis: latency and reactivation[J]. Infect Immun,2001, 69(7):4195-4201.
[4] Gatfield J, Pieters J. Essential role for cholesterol in entry of Mycobacteria into macrophages [J].Science,2000, 288(5471):1647-1650.
[5] Neyrolles O, Gould K, Gares MP, et al. Lipoprotein access to MHC Class I presentation during infection of routine macrophages with live Mycobacteria[J].Immun, 2001,166(1): 447-457.
[6] Pasula R, Wright JR, Kachel DL, et al. Surfactant protein A suppresses reactive nitrogen intermediates by alveolar macrophages in response to Mycobacterium tuberculosis[J].Clin Invest, 1999,103(4):483-490.
[7] Mahenthiralingam E, Marklund BI, Brooks LA, et al. Site-Directed Mutagenesis of the 19-Kilodalton Lipoprotein Antigen Reveals No Essential Role for the Protein in the Growth and Virulence of Mycobacterium intracellulare[J]. Infect Immun, 1998, 66(8):3626- 3634.
[8] Pieters J. Entry and survival of pathogenic mycobacteria in macrophages[J]. Microbes Infect, 2001,3(3):249-255.
[9] Anand PK, Kaul D. Downregulation of TACO gene transcription restricts mycobacterial entry/survival within human macrophages[J]. FEMS Microbiol Lett, 2005, 250(1):137-144.
[10] Voss JJD, Rutter K, Schroeder BG, et al. Iron Acquisition and Metabolism by Mycobacteria[J]. Bacteriol, 1999,181(15):4443-4451.
[11] Perez E, Samper S, Bordas Y, et al. An essential role for phoP in Mycobacterium tuberculosis virulence[J]. Mol Microbiol, 2001, 41(1):179-187.
[12] Braunstein M, Espinosa BJ, Chan J, et al. SecA2 functions in the secretion of superoxide dismutase A and in the virulence of Mycobacterium tuberculosis[J]. Mol Microbiol, 2003, 48(2): 453-464.
[13] Boom WH, Chervenal KA, Mincek MA, et al. Role of the mononuclear phagocyte as an antigen-presenting cell for human cells activated by live mycobacterium tuberculosis[J].Infect Immun,1992,60(9):3480-3488.
[14] Steffen Stenger, Modlin RL. T cell mediated immunity to Mycobacterium tuberculosis[J].Curr Opin in Microbiol,1999, 2(1):89-93.
[15] Flynn JL, Ernst JD. Immune responses in tuberculosis[J]. Curr Opin Immunol, 2000,12(4):432-436.
[16] Kurts C, Carbone FR, Barnden M, et al. CD4+T Cell Help Impairs CD8+T Cell Deletion Induced by Cross-presentation of Self-Antigens and Favors Autoimmunity[J]. Exp Med, 1997, 186 (12):2057-2062.
[17] Lazarevic V, Flynn J. CD8+T Cells in Tuberculosis[J]. Am J Respir Crit Care Med, 2002,166(8):1116-1121.
[18] Havlir DV, Ellner JJ, Chervenak KA, et al. Selective expansion of human gamma delta T cells bynon-peptide antigens and Daudi cells analyzed by TCR gene transfer[J]. Immunol,1995,154(3):998-1006.
[20] ohnson BJ, Ress SR, Willcox P, et al. Clinical and immune responses of tuberculosis patients treated with low-dose IL-2 and multidrug therapy[J]. Cytokines Mol Ther, 1995,1(3):185-196.
[21] Shingo H, Naoyuki N, Takeshi K, et al. Preliminary evaluation of soluble IL-2 receptor and type III procollagen N-terminal aminopeptide in pleural fluid for differentiating tuberculous, carcinomatous and parapneumonic pleural effusions[J]. Respirology, 2002,7(4):311-315.
[22] Gazzinell RT, Wysocka M, Hayashi S, et al. Parasite-induced IL-12 stimulates early synthesis and resistance during acute infection with Toxoplasma gondii[J]. Immunol, 1994, 153(6):2533-2543.
[23] Rhoades ER, Goopper AM, Omne IM. Chemokine response in mice infected with Mycobacterium tuberculosis[J].Infect Immun,1995,63(10):3871-3877.
[24] Cooper AM, Magram J, Ferrante J, et al. Interleukin-12 (IL-12) is crucial to the development of protective immunity in mice intravenously infected with Mycobacterium tuberculosis[J]. Exp Med,1997, 186(1):39-45.
[25] Flynn JL, Goldstein MM, Triebold KJ, et al. IL-12 increases resistance of BALB/c mice to Mycobacterium tuberculosis infection[J]. Immunol,1995,155(5):2515-2524.
[26] Das G, Sheridan S, Janeway CA. The Source of Early IFN-γ That Plays a Role in Th1 Priming[J]. Immunol, 2001,167(4): 2004-2010.
[27] Lyadova I, Yeremeev V, Majorov K, et al. An ex vivo study of T Lymphocytes recovered from the lungs of I/St mice infected with and susceptible to Mycobacterium tuberculosis[J]. Infec.Immun, 1998,66(10): 4981-4988.
[28] Orme I, Miller E, Roberts A, et al. T Lymphocytes mediating protection and cellular cytolysis during the course of Mycobacterium tuberculosis infection[J]. Immunol,1992,148(1):189-196.
[29] Cooper AM, Dalton DK, Stewart TA, et al. Disseminated tuberculosis in IFN-γ gene-disrupted mice[J]. Exp Med, 178(6): 2243-2248.
[30] Flynn JL, Chan J, Triebold KJ, et al. An essential role for Interferon-γ in resistance to Mycobacterium tuberculosis infection[J]. Exp Med.178(6):2249-2254.
[31] Lalvani A, Brookes R, Wilkinson RJ, et al. Human cytolytic and interferon gamma- secreting CD8 T lymphocytes specific for Mycobacterium tuberculosis[J]. PNAS, 1998, 95(1):270-275.
[32] Ottenhoff TH, Kumararatne D, Casanova JL. Noval human immunodeficiencies reveal the essential role of type-1 cytokines in immunity to intracellular bacteria[J]. Immunol Today, 1998,19(11):491-494.
[33] Ladel CH, Szalay G, Reidel D, et al. Interleukin-12 secretion by Mycobacterium tuberculosis infected macrophages[J].Infect Immun,1997,65(5): 1936-1938.
[34] Henderson RA, Watkins SC, Flynn JL. Activation of human dendritic cells following infection with Mycobacterium tuberculosis[J]. Immunol,1997,159 (2):635-643.
[35] Flynn JL, Goldstein MM, Chan J, et al. Tumor necrosis factor is required in the protective immune response against M.tuberculosis in mice[J].Immunity,1995, 2(6):561-572.
[36] Bean AGD, Roach DR, Briscoe H, et al. Structural deficiencies in granuloma formation in TNF gene targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection ,which is not compensated for by lymphotoxin[J]. Immunol,1999,162(6):3504-3511.
[37] Moreira AL, Tsenova L, Wang J, et al. Effect of cytokine modulation by thalidomide on the gramomatous response in murine tuberculosis[J].Tubercle Lung Disease, 1997,78(1):47-55.
[38] Hodge DJ, Marino MW, Horton MR, et al. Inhibition of interferon induced interleukin 12 production:a potential mechanism for the anti-inflammatory activities of tumor necrosis factor[J].PNAS, 1998,95(23): 13806-13811.
[39] Fenhalls G, Wong A, Bezuidenhout J, et al. In situ production of gamma interferon,interleukin-4,and tumor necrosis factor alpha mRNA in human lung tuberculous granuloma[J].Infect Immun, 2000,68(5):2827- 2836.
[40] Gong J, Zhang M, Modlin R, et al. Interleukin-10 downregulates Mycobacterium tuberculosis-induced Th1 responses and CTLA4 expression[J]. Infect Immun, 1996,64(3):913-918.
[41] Rojas M, Olivier M, Gros P, et al. TNFα and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages[J].Immunol, 1999,162(10):6122-6131.
[42] Ladel R, Miyajima A, Mee PJ, et al. Lethal tuberculosis in interleukin-6-deficient mutant mice[J].Blood,1996,88(7):2458-2464.
[43] Reed JA, Ikegami M, Robb L, et al. Cytokine gene expression by cultures of human lymphocytes with autologous Mycobacterium tuberculosis-infected monocytes[J].AmJ Physiol Lung Cell Mol Physiol,2000,278(6):1164-1171.
[44] Johnson BJ, Ress SR, Willcox P, et al. Clinical and immune responses of tuberculosis patients treated with low-dose IL-2 and multidrug therapy[J]. Cytokines Mol Ther, 1995,1(3):185-196.
[45] Fine PE. Variation in protection by BCG: implications of and for heterologous immunity [J].Lancet,1995,346(8986):1339-1345.
[46] Rook GA, Dheda K, ZumLa A. Immune responses to tuberculosis in developing countries: implications for new vaccines[J]. Nat Rev Immunol 2005,5(8):661-667.
[47] Brooks JV, Frank AA, Keen MA, et al. Boosting vaccine for tuberculosis[J]. Infect Immu -n,2001,69(4):2714-2717.
[48] van Rie A, Warren R, Richardson M, et al. Exogenous Reinfection as a Cause of Recurrent Tuberculosis after Curative Treatment[J]. N Engl J Med,1999,341(16):1174- 1179.
[49] Behr MA, Wilon MA, Gill WP, et al. Comparative genomics of BCG vaccines By whole genome DNA microarry[J].Science,1999,284(5419):1520-1523.
[50] Lewis KN, Liao R, Guinn KM, et al. Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guerin attenuation[J]. Infect Dis, 2003, 187(1):117-123.
[51] Orme IM, Collins FM. Efficacy of Mycobacterium bovis BCG vaccination in mice undergoing prior pulmonary infection with atypical mycobacteria. Infect[J]. Immun, 1984,44(1):28-32.
[52] Hubbard RD, Flory CM, FM Collins. Immunization of mice with mycobacterial culture filtrate proteins[J].Clin Exp Immunol, 1992,7(1):94-98.
[53] Sonnenberg MG, Belisle JT. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N- terminal amino acid sequencing, and electrospray mass spectr- ometry[J].Infect Immun,1997,65(1):4515-4524.
[54] Andersen P, Andersen AB, Sorensen AL, et al. Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice[J]. Immunol, 1995,154(7):3359-3372.
[55] Li ZM, Howard A, Kelley C, et al. Immunogenicity of DNA vaccines expressing tuberculosis proteinsfused to tissue plasminogen activator signal sequences[J]. Infect Immun,1999,67(9):4780-4786.
[56] Zhu XJ, Venkataprasad N, Thangaraj HS, et al. Functions and specificity of T ells following nucleic acid vaccination of mice against Mycobacterium tuberculosis[J]. Immunol,1997, 158(12):5921-5926.
[57] Erb KJ, Kirman J, Woodfield L, et al. Identification of potential CD8+T-cell epitopes of the 19kua and AhpC proteins from Mycobacterium tuberculosis. No evidence for CD8+ T-cell priming against the identified peptides after DNA-vaccination of mice[J]. Vaccine,1998,16(7):692-697.
[58] Anderson P, Askgaard D, Gottschau A, et al. Identification of immunodominant antigens during infection with Mycobacterium tuberculosis[J]. Scand J Immunol,1992,36(6):823-831.
[59] Olsen AW, Laurens AH, Pinxteren V, et al. Protection of Mice with a Tuberculosis Subunit Vaccine Based on a Fusion Protein of Antigen 85B and ESAT-6[J]. Infect Immun,2001,69(5):2773-2778.
[60] Horwitz MA, Lee BW, Dillon BJ, et al. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis[J]. PNAS,1995,92(5):1530-1534.
[61] Lindblad EB, Elhay MJ, Silva R, et al. Adjuvant modulation of immune responses to tuberculosis subunit vaccines.Infect[J]. Immun, 1997,65(2): 623-629.
[62] Sinha RK, Khuller GK. The protective efficacy of a liposomal encapsulated 30 kua secretory protein of Mycobacterium tuberculosis H37Ra against tuberculosis in mice[J].Immunol Cell Biol,1997,75(5): 461-466.
[63] Vordermeier HM, Coombes AG, Jenkins P, et al. Synthetic delivery system for tuberculosis vaccines: immunological evaluation of the M. tuberculosis 38 kua protein entrapped in biodegradable PLG microparticles[J].Vaccine,1995,13(16):1576-582.
[64] Stenger S. An antimicrobial activity of cytolytic T cells mediated by granulysin[J]. Science,1998,282(5386):121-125.
[65] Lowrie DB, Silva CL. Enhancement of immunocompetence in tuberculosis by DNA vaccination [J].Vaccine, 2000(18):1712-1716.
[66] 范雄林, 李元, 徐志凯. 结核分支杆菌基因疫苗研究进展[J].细胞与分子免疫学杂志, 2001,17(4):121-123.
[67] Morris S, Kelley C, Howard A, et al. The immunogenicity of single and combination DNA vaccines against tuberculosis[J].Vaccine,2000,18(20): 2155-2163.
[68] Kamath AT, Feng CG, Macdonald M, et al. Differential Protective Efficacy of DNA Vaccines Expressing Secreted Proteins of Mycobacterium tuberculosis[J]. Infect Immun, 1999, 67(4):1702-1707.
[69] 师长宏, 范雄林, 徐志凯, 等.结核分支杆菌分泌蛋白 Ag85B-ESAT-6 的融合表达及纯化[J].中华结核和呼吸杂志,2004,(2): 89-92.
[70] 师长宏, 范雄林, 徐志凯, 等. 融合表达 Ag85B-ESAT-6 的结核分支杆菌真核表达载体的构建及其免疫原性[J].第四军医大学学报,2004,25(2):121-124.
[71] Bonato VL, Lima VM, Tascon RE, et al. Identification and characterization of protective T cells in hsp65 DNA-vaccined and M. tuberculosis infected mice[J]. Infect Immun, 1998,66(5):169-175.
[72] Tacson RE, Colston MJ, Ragno S, et al. Vaccination against tuberculosis by DNA injection[J].Nat Med,1996,2(8):888- 892.
[73] Kumar V, Sercarz E. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine[J]. NatMed,1996,2(8):857-859.
[74] Ulmer JB, Liu MA, Montgomery DL, et al. Expression and immunogenicity of M.tuberculosis antigen 85 by DNA vaccination[J]. Vaccine,1997,15(8):792-794.
[75] Lozes E, Huygen K, Content J, et al. Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex[J].Vaccine,1997,15(8):830-833.
[76] Denis O, Tanghe A, Palfliet K, et al. Vaccination with plasmid DNA encoding mycobacterial antigen 85A stimulates a CD4+ and CD8+ T-cell epitopic repertoire broader than that stimulated by M. tuberculosis H37Rv infection[J]. Infect Immun, 1998,66(4):1527-1533.
[77] Baldwin SL, D’SOUZA CD, Orme IM, et al. Immunogenicity and protective efficacy of DNA vaccines encoding secreted and non-secreted forms of Mycobacterium tuberculosis Ag85A[J]. Tuber Lung Dis,1999,79(4):251-259.
[78] Dillon DC, Alderson MR, Day CH, et al. Molecular Characterization and human T- cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family[J]. Infect Immun,1999,67(4):2941-2950.
[79] Lefevre P, Denis O, De Wit L, et al. Cloning of the genge encoding a 22-kilodalton cell surface antigen of Mycobacterium bovis BCG and analysis of its potential for DNA vaccination against tuberculosis[J]. Infect Immun, 2000,68(3):1040-1047.
[80] Tanghe A, Lefevre P, Denis O, et al. Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors[J]. Immunol,1999,162(2):1113-1119.
[81] Chambers MA, Vordermeier H, Whelan A, et al. Vaccination of mice and cattle with plasmid DNA encoding the Mycobacterium bovis antigen MPB83[J].Clin Infect Dis,2000,30(Suppl3): S283-287.
[82] Grover A, Ahmed MF, Verma I, et al. Expression and purification of the Mycobacterium tuberculosis complex-restricted antigen CFP21 to study its immunoprophylactic potential in mouse model[J]. Protein Expr Purif, 2006, 48(2): 274-280.
[83] Hervas-Stubbs S, Majlessi L, et al. High Frequency of CD4+ T Cells Specific for the TB10.4 Protein Correlates with Protection against Mycobacterium tuberculosis Infection[J]. Infect. Immun., 2006,74(6):3396-3407.
[84] Coler RN, Campos-Neto A, Ovendale P, et al. Vaccination with the T Cell Antigen Mtb 8.4 Protects Against Challenge with Mycobacterium tuberculosis[J]. Immunol, 2001, 166(10):6227-6235.
[85] Ha SJ, Jeon BY, Kim SC, et al. Therapeutic effect of DNA vaccines combined with chemotherapy in a latent infection model after aerosol infection of mice with Mycobacterium tuberculosis[J]. Gene Ther 2003;10(18):1592-1599.
[86] Ha SJ, Jeon BY, Youn JI, et al. Protective effect of DNA vaccine during chemotherapy on reactivation and reinfection of Mycobacterium tuberculosis[J]. Gene Ther 2005,12(7):634–638.
[87] Silva CL, Bonato VLD, Coelho-Castelo AAM, et al. Immunotherapy with plasmid DNA encoding mycobacterial hsp65 in association with chemotherapy is a more rapid and efficient form of treatment for tuberculosis in mice[J]. Gene Ther 2005;12(3):281-287.
[88] Nuermberger E, Tyagi S, Williams KN, et al. Rifapentine, Moxifloxacin, or DNA Vaccine Improves Treatment of Latent Tuberculosis in a Mouse Model[J]. Am. J Respir Crit Care Med, 2005,172(11): 1452-1456.
[89] Zhu X, Venkataprasad N, Ivanyi J, et al.Vaccination with recombinant vaccinia viruses protects miceagainst Mycobacterium tuberculosis infection[J].Immunology,1997,92(1):6-9.
[90] Hess J, Grode L, Hellwig J, et al. Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kua antigen of Mycobacterium bovis BCG[J]. FEMS Immunol Med Microbiol, 2000, 27(4):283-289.
[91] Chan J, Kaufmann S. Immune mechanisms of protection in tuberculosis pathogenesis, protection and control.Ed[A]. American Society of Microbiology[C]. Washington, D.C: BR Bloom, 1994,9389-9415.
[92] Bange FC, Brown AM, Jacobs WR. Leucine auxotrophy restricts growth of mycobacterium bovis BCG in macrophages[J]. Infect Immun,1996,64(5):1794- 1799.
[93] Smith DA, Parish T, Stoker NG, et al. Characterization of auxotrophic mutants of mycobacterium tuberculosis and their potential as vaccine candidates[J]. Infect Immun,2001,69(2):1142-1150.
[94] Jackson M, Phalen SW, Lagranderie M, et al. Persistence and protective efficacy of mycobacterium tuberculosis auxotroph vaccine[J]. Infect Immun, 1999,67(6): 2867-2873.
[95] Horwitz MA, Harth G, Dillon BJ, et al. Recombinant bacillus Calmette-Guerin(BCG) vaccines expressing the Mycobacterium tuberculosis 30-kua major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model[J].PNAS,2000,97(25):13853-13858.
[96] Bao L, Chen W, Zhang H, et al. Virulence, immunogenicity, and protective efficacy of two recombinant Mycobacterium bovis Bacillus Calmette-Guerin strains expressing the antigen ESAT6 from Mycobacterium tuberculosis[J]. Infect Immun,2003,71(4): 1656-1661.
[97] Hess J, Miko D, Catic A, et al. Mycobacterium bovis Bacille Calmette Guerin strains secreting listeriolysin of Listeria monocytogenes[J].PNAS,1998,95(9):5299-5304.
[98] 赵宝华, 张莉, 石振华.基因重组卡介苗的优点及应用[J].生物学通报. 2002,37(1):18-20.
[99] Hart HG, Lee BY, Horwitz MA. High level heterologous expression and secretion in rapidly growing nonpat hogenic Mycobacteria of four major Mycobacterium tuberculosis ext racellular proteins considered to be leading vaccine candidates and drug targets[J]. Infect Immun,1997,65 (6):2321-2328.
[100] Falcone V, Bassey E, Jacobs W Jr, et al. The immunogenicity of recombinant mycobacterium smegmatis bearing BCG genes[J]. Microbiology, 1995,141(pt5):1239-1245.
[101] 胡佳杰, 文学明, 肖爱清, 等. 重组 BCG-hsp70 疫苗有关毒性实验研究[J]. 广西预防医学,2002,8(4):233-235.
[102] 胡佳杰, 文学明, 肖爱清, 等. 重组 M.S-Sj26GST 疫苗有关毒性实验研究[J].华南预防医学,2002,28(5):44-45.
[103] Stenger S, Mazzaccaro RJ, Uyemura K, et al. Differential effects of cytolytic T-cell subsets on intracellular infection[J]. Science,1997,276(5319): 1684-1687.
[104] Andersen P, Askgaard D, Gottschau A. Identification of immunod ominant antigens during infection with Mycobacterium tuberculosis[J].Scand J Immunol, 1992,36(2):823-828.
[105] Kaufman SHE. The cellular immune response to heat shock proteins[J]. Experientia, 199 2,48(3):640-645.
[106] Charo J, Geluk A, Sundback M, et al. The identification of a comLnon pathogen-specfic HLA class I A*0201-restricted cytolytic T cell epitope encoded within the heat shock protein 65[J].Eur J Immunol, 2001,31(12):3602- 3611.
[107] Ragno S, Colston MJ, Lowrie DB, et al. Protection of rats from adjuvant arthritis by immunization with naked DNA encoding for mycobacterial heat shock protein 65[J]. Arthritis Rheum,1997, 40(2):277-283.
[108] [108] Turner OC, Roberts AD, Fran k AA, et al. Lack of protection in mice an d necrotizing bronchointerstitial pneumonia bronchiolitis in sninea pigs immunized with vaccines directed against the HSP65 molecular of Mycobacterium tuberculosis[J]. Infect Immun,2000,68(6):3674-3679.
[109] 刘思国,王春来,彭永刚,等.牛分支杆菌热休克蛋白 65 基因的分子克隆和表达[J]. 中国预防兽医学报,2004,26(5):336-271.
[110] Shinnick TM. The 65-kilodalton antigen of Mycobacterium tuberculosis[J].Bacteriol, 1987, 169 (3):1080-1088.
[111] H.Martin Vordermeier, Douglas B. Lowrie, R. Glyn Hewinson. Improved immunogenicity of DNA vaccination with mycobacterial HSP65 against bovine tuberculosis by protein boosting[J].Veterinary Microbiology,2003, (93) : 349–359.
[112] Yang BF, Zhao HL, Xue C, et al.Recombinant heat shock protein 65 carrying hepatitis B core antigen induces HBcAg-specific CTL response[J]. Vaccine.,2007, 25(22):4478-86.
[113] Lowrie DB, Tascon RE, Bonato VL, et al. Therapy of tuberculosis in mice by DNA vaccination[J]. Nature, 1999, 400(6741): 269-271.
[114] 居巍, 刘君炎, 庄玉辉, 等. 结核杆菌pCHSP65真核表达载体的构建及其DNA免疫实验[J].中国免疫学杂志, 2003,19(1):12-15.
[115] J.萨姆布鲁克, EF.弗里奇, T.曼尼阿蒂斯. 分子克隆实验指南[M]. 北京: 科学出版社,1996.49-55.
[116] Silva C L, Lowrie D B. A single mycobacterial protein (hsp65) expressed by a transgenic antigen-presenting cell vaccinates mice against tuberculosis[J] . Immunology, 1994, 82:244.
[117] Lowrie D B, Silva C L, Colston MJ, et al. Protection against by a plasmid DNA vaccine [J]. Vaccine, 1997, 15:834.
[118] Schirmbeck R, Melber K, Kuhrober A, et al. Immunization with soluble hepatitis B virus surface protein elicits murine H-2 class I-restricted CD8+ cytotoxic T lymphocyte responses in vivo[J]. Immunol, 1994,152(3):1110-1119.
[119] Stewart MJ, Plautz GE, Del Buono L, et al. Gene transfer in vivo with DNA-liposome complexes: safety and acute toxicity in mice[J]. Hum Gene Ther, 1992,3(3):267-275.
[120] Elhay H,Martin J,Ersen et al.Immunological requirements for a subunit vaccine against tuberculosis [J].Immunology and Cell Biology.1997,75(6):595-603.
[121] 薛莹.结核分支杆菌Rpf样蛋白生物学及免疫学活性的研究[D].第四军医大学博士论文.2005年.