结核分枝杆菌基因疫苗的构建及其免疫学特性的初步研究
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摘要
结核病（tuberculosis，TB）是由结核分枝杆菌（Mycobacterium
tuberculosis，MTB）所致以呼吸道为主的慢性传染病。目前全球有1/3的人
口感染MTB，每年有1000万的新发病人和300万的病人死亡，表明TB
仍是影响全球人类健康的一个主要公共卫生问题。近年来，耐药结核分
枝杆菌的流行、与HIV的合并感染以及人口流动的增加等因素，进一步
加剧了TB对人类的威胁。卡介苗（BCG）是目前TB唯一的预防性疫苗，
在发展中国家广泛应用，但其保护效果不稳定。另外，BCG接种干扰了
利用PPD对MTB感染的诊断，接种免疫缺陷病人可能导致播散性感染
等缺陷。鉴于BCG自身的不足，以及当前TB流行的严重性，研究更有
效的疫苗具有重要意义。
基因免疫在包括TB的多种传染病的动物模型中可引起体液和细胞
免疫应答。利用质粒DNA真核表达载体，已经有24个MTB抗原被表
达，这些抗原主要来自MTB培养滤液蛋白（CFP）。其中有几个蛋白在
动物模型中显示了较高的保护性，如Ag85复合体、ESAT6、MPT64、
Hsp65和PstS3等。因而基因免疫可能是有希望的用于防治TB的一种有
效方法。
MTB CFP中的蛋白质种类高达800多种。目前从中鉴定保护性抗原
的方法主要是：利用生物化学的方法分离蛋白，以刺激淋巴细胞分泌细
胞因子；利用TB病人血清或MTB感染鼠血清筛检MTB表达文库。
第四军医大学博十学位论文
MTB基因疫茵的研究也可作为一种潜在的研究方法，在体内用于保护性
抗原的筛选鉴定。
为了TB新型疫苗的研究，以及建立快速、有效、廉价的基因免疫
筛选保护性抗原的模式，我们采用 PCR的方法从 MTB H37Ra株基因组
中，扩增出分泌蛋白Ag85B的成熟蛋白的编码基因，用限制性内切酶消
化后，插入克隆载体 pUC中。经酶切鉴定与序列测定证实后，以亚克
隆法构建于真核表达载体pcDNA3的相应酶切位点，再用酶切鉴定证实
相应片段的正确插入。该重组子命名为pTB30m。所测定的序列用Blast
程序检索，表明 MTB H37Ra株Ag85B蛋白的编码基因与结核分枝杆菌
复合体和非结核分枝杆菌相应蛋白的基因高度同源，该序列登录入
Genbank（accession number：AF198032）。将 pTB30m以电穿子法转染 CHO
细胞，并以RTFCR法检测转染细胞中Ag85B特异的mRNA的表达，
初步证实了该质粒可在体外真核细胞中表达：另用该质粒肌注免疫
BALB／c和C57BL历小鼠，ELISA法检测免疫小鼠的体液免疫应答效果，
证实了该质粒可在体内表达。两种小鼠血清中Ag85B特异性抗体滴度分
别约为1：60和1：80。
为了检测pTB30m质粒免疫小鼠引起的细胞免疫反应，基因免疫完
成后第六周，分离免疫小鼠的脾淋巴细胞，体外用抗原再刺激，生物学
方法检测 IFN Y产生水平和 MTT法检测淋巴细胞增殖反应。pTB30m质
粒免疫的 BALB／c和 C57BU6小鼠脾细胞分泌的 IFN Y的量分别达到
1360ng／ml和 1965pg／ml，生理盐水和空质粒对照组均低于 80 pg／ml；脾
脏淋巴细胞增殖显著，刺激指数分别为3．34和4．23，生理盐水和空质粒
对照组分别为 1．39和 1．sl。
为进一步评价基因免疫的保护性，用 MTB H37RV毒株 SX fCFU
经小鼠尾静脉攻击感染基因免疫的BALB／c ，J’鼠，4W后，脾脏细菌负荷
计数。与生理盐水对照组相比，pTB30m质粒免疫BALB儿 ’J’鼠的脾脏
细菌负荷减少（0．645logl。）CFU（t＝8．584，P＜0刀05，i＝3），而空质粒对照组
仅减少（0刀57log；。）CFU（t＝l．334，P＞0．20，n＝3）。为了证实 T细胞在基因
免疫中的作用，用尼龙毛柱分离基因免疫BALB／c小鼠的T淋巴细胞，
4
第四军医人学博士学位论文
每只 5 XIO‘过继免疫正常 BALB止 ，J’鼠，同时用 MTB ＄株 10’CFU经
小鼠尾静脉攻击感染，4w后，脾脏细菌负荷计数。pTB30m质粒免疫小
鼠的T细胞过继免疫对攻击感染后抗MTB在脾脏中增殖有一定程度的
保护作用0＜0刀5，r3．534，n＝3人与生理盐水阴性对照组相比，脾脏细菌
负荷减少O．285loglOCFU。而质粒空白对照组脾脏细菌负荷减少
（0．182logl。）CFU（0．10＜P＜0二0，t＝l石37，11＝3）。
综上所述，采用编码 MTB H37Ra株 Ag85B成熟蛋白基因的真核质
粒pTB30m基因免疫，是产生抗原特异性的体液免疫反应和保护性细胞
Tuberculosis(TB) is a chronic respiratory infectious disease caused by the
pathogen Mycobacterium fuberculosis. TB remains an important global public
health problem, because about one-third of the world's populations are infected
with the M. tuberculosis and there are 10 million new cases and 3 million
deaths annually, in recent years, such factors as increasing frequency of
drug-resistance M tuberculosis isolates and incidence of HI V-associated
tuberculosis and increasement of population movement, have aggravated
further the opportunity of people worldwide face to the threat of TB.
Mycobacterium bovis BCG is the only tuberculosis vaccine available for
human use in many developing countries, yet its protection efficacy varies
greatly. Another limitation is that it interrupts the diagnosis of TB by using
purified protein derivative (PPD). Furthermore, the live BCG vaccine
represents a potential health risk to immunocompromized individuals. It should
be very important to develop a more effective vaccine of TB.
It has been proved that the administration of DNA vaccines could
generate both hwnoral and cell-mediated immune responses in many animal
models of infectious diseases including TB. 24 mycobacterial antigens
6
mostly from cultural filtration proteins(CFP) of Mtuberculosis have been
expressed using plasmid DNA eukaryotic vector. Among these, several
antigens have showed higher protection efficacy in animal models such as
Ag85 complex, ESAT6, MPT64, Hsp65 and PstS3, etc. Thus, DNA
vaccination could be a promising effective solution to protect against TB.
It has been showed that over 800 proteins result in the CFP of
Mtuberculosis. Important methods used to identify protective antigens from
CFP include, that proteins separated by biochemistry methods could be used
to stimulate lymphocytes and detect kinds and quantity of cytokines secreted
by lymphocytes; and that sera of patients with TB or mice infected with
Mtuberculosis could be used to screen expression library of Mtuberculosis.
The development of DNA vaccine of Mtuberculosis could be a potential way
to identify protective antigens in vivo.
In this paper, we want to develop new vaccine against TB and search for
a rapid, effective and inexpensive model of DNA vaccination to screen
protective antigens. The gene encoding the Ag85B mature form with two
amino acids of signal sequence from genome of M tuberculosis H37Ra strain
was amplified by PCR., and inserted it into cloning vector pUC 19 after
restriction endonuclease digestion. Gene fragment encoding Ag85B mature
protein was correctly inserted into the vector as confirmed by partial
nucleotide sequencing and restriction endonuclease digestion, and then was
subcloned to sites cut with HindIIl plus EcoR I of eukaryotic expression
vector pcDNA3(Invitrogen Corp.) under the control of the CMV promoter.
The cloning gene was correctly inserted into the vector pcDNA3 as also
confirmed by restriction endonuclease digestion. This construction was called
pTB3Om. Sequence of Ag85B mature protein was compared by Blast
homological search via GenBank. It shows that nucleotide sequence of
Ag85B mature protein of Mtuberculosis I-137Ra strain has the highest degree
of homology with that of corresponds of M tuberculosis complexes and
7
higher degree of similarity with that of corresponding proteins of
non-M tuberculosis. Nucleotide sequence
tuberculosis，MTB）所致以呼吸道为主的慢性传染病。目前全球有1/3的人
口感染MTB，每年有1000万的新发病人和300万的病人死亡，表明TB
仍是影响全球人类健康的一个主要公共卫生问题。近年来，耐药结核分
枝杆菌的流行、与HIV的合并感染以及人口流动的增加等因素，进一步
加剧了TB对人类的威胁。卡介苗（BCG）是目前TB唯一的预防性疫苗，
在发展中国家广泛应用，但其保护效果不稳定。另外，BCG接种干扰了
利用PPD对MTB感染的诊断，接种免疫缺陷病人可能导致播散性感染
等缺陷。鉴于BCG自身的不足，以及当前TB流行的严重性，研究更有
效的疫苗具有重要意义。
基因免疫在包括TB的多种传染病的动物模型中可引起体液和细胞
免疫应答。利用质粒DNA真核表达载体，已经有24个MTB抗原被表
达，这些抗原主要来自MTB培养滤液蛋白（CFP）。其中有几个蛋白在
动物模型中显示了较高的保护性，如Ag85复合体、ESAT6、MPT64、
Hsp65和PstS3等。因而基因免疫可能是有希望的用于防治TB的一种有
效方法。
MTB CFP中的蛋白质种类高达800多种。目前从中鉴定保护性抗原
的方法主要是：利用生物化学的方法分离蛋白，以刺激淋巴细胞分泌细
胞因子；利用TB病人血清或MTB感染鼠血清筛检MTB表达文库。
第四军医大学博十学位论文
MTB基因疫茵的研究也可作为一种潜在的研究方法，在体内用于保护性
抗原的筛选鉴定。
为了TB新型疫苗的研究，以及建立快速、有效、廉价的基因免疫
筛选保护性抗原的模式，我们采用 PCR的方法从 MTB H37Ra株基因组
中，扩增出分泌蛋白Ag85B的成熟蛋白的编码基因，用限制性内切酶消
化后，插入克隆载体 pUC中。经酶切鉴定与序列测定证实后，以亚克
隆法构建于真核表达载体pcDNA3的相应酶切位点，再用酶切鉴定证实
相应片段的正确插入。该重组子命名为pTB30m。所测定的序列用Blast
程序检索，表明 MTB H37Ra株Ag85B蛋白的编码基因与结核分枝杆菌
复合体和非结核分枝杆菌相应蛋白的基因高度同源，该序列登录入
Genbank（accession number：AF198032）。将 pTB30m以电穿子法转染 CHO
细胞，并以RTFCR法检测转染细胞中Ag85B特异的mRNA的表达，
初步证实了该质粒可在体外真核细胞中表达：另用该质粒肌注免疫
BALB／c和C57BL历小鼠，ELISA法检测免疫小鼠的体液免疫应答效果，
证实了该质粒可在体内表达。两种小鼠血清中Ag85B特异性抗体滴度分
别约为1：60和1：80。
为了检测pTB30m质粒免疫小鼠引起的细胞免疫反应，基因免疫完
成后第六周，分离免疫小鼠的脾淋巴细胞，体外用抗原再刺激，生物学
方法检测 IFN Y产生水平和 MTT法检测淋巴细胞增殖反应。pTB30m质
粒免疫的 BALB／c和 C57BU6小鼠脾细胞分泌的 IFN Y的量分别达到
1360ng／ml和 1965pg／ml，生理盐水和空质粒对照组均低于 80 pg／ml；脾
脏淋巴细胞增殖显著，刺激指数分别为3．34和4．23，生理盐水和空质粒
对照组分别为 1．39和 1．sl。
为进一步评价基因免疫的保护性，用 MTB H37RV毒株 SX fCFU
经小鼠尾静脉攻击感染基因免疫的BALB／c ，J’鼠，4W后，脾脏细菌负荷
计数。与生理盐水对照组相比，pTB30m质粒免疫BALB儿 ’J’鼠的脾脏
细菌负荷减少（0．645logl。）CFU（t＝8．584，P＜0刀05，i＝3），而空质粒对照组
仅减少（0刀57log；。）CFU（t＝l．334，P＞0．20，n＝3）。为了证实 T细胞在基因
免疫中的作用，用尼龙毛柱分离基因免疫BALB／c小鼠的T淋巴细胞，
4
第四军医人学博士学位论文
每只 5 XIO‘过继免疫正常 BALB止 ，J’鼠，同时用 MTB ＄株 10’CFU经
小鼠尾静脉攻击感染，4w后，脾脏细菌负荷计数。pTB30m质粒免疫小
鼠的T细胞过继免疫对攻击感染后抗MTB在脾脏中增殖有一定程度的
保护作用0＜0刀5，r3．534，n＝3人与生理盐水阴性对照组相比，脾脏细菌
负荷减少O．285loglOCFU。而质粒空白对照组脾脏细菌负荷减少
（0．182logl。）CFU（0．10＜P＜0二0，t＝l石37，11＝3）。
综上所述，采用编码 MTB H37Ra株 Ag85B成熟蛋白基因的真核质
粒pTB30m基因免疫，是产生抗原特异性的体液免疫反应和保护性细胞
Tuberculosis(TB) is a chronic respiratory infectious disease caused by the
pathogen Mycobacterium fuberculosis. TB remains an important global public
health problem, because about one-third of the world's populations are infected
with the M. tuberculosis and there are 10 million new cases and 3 million
deaths annually, in recent years, such factors as increasing frequency of
drug-resistance M tuberculosis isolates and incidence of HI V-associated
tuberculosis and increasement of population movement, have aggravated
further the opportunity of people worldwide face to the threat of TB.
Mycobacterium bovis BCG is the only tuberculosis vaccine available for
human use in many developing countries, yet its protection efficacy varies
greatly. Another limitation is that it interrupts the diagnosis of TB by using
purified protein derivative (PPD). Furthermore, the live BCG vaccine
represents a potential health risk to immunocompromized individuals. It should
be very important to develop a more effective vaccine of TB.
It has been proved that the administration of DNA vaccines could
generate both hwnoral and cell-mediated immune responses in many animal
models of infectious diseases including TB. 24 mycobacterial antigens
6
mostly from cultural filtration proteins(CFP) of Mtuberculosis have been
expressed using plasmid DNA eukaryotic vector. Among these, several
antigens have showed higher protection efficacy in animal models such as
Ag85 complex, ESAT6, MPT64, Hsp65 and PstS3, etc. Thus, DNA
vaccination could be a promising effective solution to protect against TB.
It has been showed that over 800 proteins result in the CFP of
Mtuberculosis. Important methods used to identify protective antigens from
CFP include, that proteins separated by biochemistry methods could be used
to stimulate lymphocytes and detect kinds and quantity of cytokines secreted
by lymphocytes; and that sera of patients with TB or mice infected with
Mtuberculosis could be used to screen expression library of Mtuberculosis.
The development of DNA vaccine of Mtuberculosis could be a potential way
to identify protective antigens in vivo.
In this paper, we want to develop new vaccine against TB and search for
a rapid, effective and inexpensive model of DNA vaccination to screen
protective antigens. The gene encoding the Ag85B mature form with two
amino acids of signal sequence from genome of M tuberculosis H37Ra strain
was amplified by PCR., and inserted it into cloning vector pUC 19 after
restriction endonuclease digestion. Gene fragment encoding Ag85B mature
protein was correctly inserted into the vector as confirmed by partial
nucleotide sequencing and restriction endonuclease digestion, and then was
subcloned to sites cut with HindIIl plus EcoR I of eukaryotic expression
vector pcDNA3(Invitrogen Corp.) under the control of the CMV promoter.
The cloning gene was correctly inserted into the vector pcDNA3 as also
confirmed by restriction endonuclease digestion. This construction was called
pTB3Om. Sequence of Ag85B mature protein was compared by Blast
homological search via GenBank. It shows that nucleotide sequence of
Ag85B mature protein of Mtuberculosis I-137Ra strain has the highest degree
of homology with that of corresponds of M tuberculosis complexes and
7
higher degree of similarity with that of corresponding proteins of
non-M tuberculosis. Nucleotide sequence
引文
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36 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. Infect Immun, 1998,66:1527-1533．
37 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. Tuber Lung Dis, 1999,79:251-259．
38 Kamath AT, Feng CG, Macdonald M, et al. Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect Immun, 1999,67:1702-1707．
39 Li ZM, Howard A, Kelley C, et al. Immunogenicity of DNA vaccines expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences. Infect Immun,1999,67:4780-4786．
40 Morris S, Kelley C, Howard A, et al. The immunogenicity of single and combination DNA vaccines against tubeculosis. Vaccine,2000,18:2155-2163．
41 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. Infect Immun, 1999,67:2941-2950．
42 Zhu XJ, Venkataprasad N, Thangaraj HS, et al. Functins and specificity of T ells following nucleic acid vaccination of mice against Mycobacterium tuberculosis. J Immunol, 1997,158:5921-5926．
43 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. Infect Immun, 2000,68:1040-1047．
44 Erb KJ, Kirman J, Woodfield L, et al Identification of potential CD8+ T-cell epitopes of the 19kda and AhpC proteins from Mycobacterium tuberculosis. No evidence for CD8+T-cell priming against the identified peptides after DNA-vaccination of mice. Vaccine,1998,16:692-697．
45 Tanghe A, Lefevre P, Denis O, et al. Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors.J Immunol, 1999,162:1113-1119．
46 Chambers MA, Vordermeier H, Whelan A, et al. Vaccination of mice and cattle with plasmid DNA encoding the Mycobacterium bovis antigen MPB83． Clin Infect Dis,2000 ,30 (Suppl 3) :S283-287．
47 Tanghe A, Denis O, Lambrecht B, et al. Tuberculosis DNA vaccine encoding Ag85A is immunogenic and protective when administered by intramuscular needle injection but not by epidermal gene gun bombardment. Infect Immun, 2000,68(7) :3854-3860．
48 Kamath AT, Hanke T,Briscoe H, et al. Co-immunization with DNA vaccines expressing granulocyte-macrophage colony stimulating factor and mycobacterial secreted proteins enhances T-cell immunity, but not protective efficacy against Mycobacterium tuberculosis. Immunnol, 1999, 96: 511-516．
49 Chow YH, Chiang BL,Lee YL, et al. Development of Th1 and Th2 populations and the nature of immune responses to Hepatitis B virus DNA vaccines can be modulated by codelivery of various cytokine genes.J Immunol, 1998,160:1320-1329．
50 Tanghe A, D'Souza S, Rosseels V,et al. Improved immunogenicity and protective efficacy of a tuberculosis DNA vaccine encoding Ag85 by protein boosting. Infect Immun, 2001,69(5) :3041-3047．
51 Lowrie DB, Tascon RE, Bonato VLD, et al. Therapy of tuberculosis in mice by DNA vaccination. Nature, 1999,400:269-271．
52 Jungblut PR, Schaible UE, Mollenkopf HJ, et al. Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens. Mol-Microbiol, 1999,33: 1103-1117．
53 Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of
Mycobacterium tuberculosis from the complete genome sequence. Nature, 1998,393:537-544.
54 Walsh GP, et al. The Philippine cynomolgus monkey (Macaca fasicularis) provides a new nonhuman primate model of tuberculosis that resembles human disease. Nature Med. 1996,2:430-436.
55 Barry MA, Lai WC, Johnston SA. Protection against mycoplasma infection using expression-library immunization. Nature, 1995,377:632-635.
56 Sykes KF, Johnston SA. Linear expression elements: a rapid, in vivo, method to screen for gene functions. Nat Biotechnol, 1999,17(4):355-359.
57 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press, 1989.
58 Harth G, Lee BY, Horwitz MA. High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets. Infect Immun, 1997,65(6):2321-2328.
59 夏云，雷二庆，王槐春。Internet实用技术与生物医学应用。北京：军事医学科学出版社，1997。
60 孙志贤。现代生物化学理论与研究技术。北京：军事医学科学出版社，1995。
61 Tang DC, Devit M, Johnston SA, et al. Genetic immunization is a simple method for eliciting an immune response. Nature, 1992,356:152-154.
62 Tighe H, Corr M, Roman M, et al. Gene vaccination: plasmid DNA is more than just a blueprint. Immunol today, 1998,19(2):89-97.
63 Wiker HG, Harboe M. The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol Rev, 1992,56(4):648-661.
64 Torres M, Herrera T, Villareal H, et al. Cytokine profiles for peripheral blood lymphocytes from patients with active pulmonary tuberculosis and healthy household contacts in response to the 30-kilodalton antigen of Mycobacterium tuberculosis. Infect Immun, 1998,66(1):176-180.
65 Belisle JT, Vissa VD, Sievert T, et al. Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science 1997,276:1420-1422.
66 Silver RF, Wallis RS, Ellner JJ. Mapping of T cell epitopes of the 30-kDa antigen of Mycobacterium boris Bacillus Calmette-Guerin in purified protein derivative(PPD)- positve individuals. J Immunol., 1995,154:4665-4674.
67 Geluk A, van Meijgaarden KE, Franken KL, et al. Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A~*0201-restricted CD8+ T cells in HLA-transgenic mice and humans. J Immunol, 2000,165(11):6463-6471.
68 李国利，庄玉辉。分枝杆菌。北京：人民军医出版社。1985。
69 孙卫民，王惠琴。细胞因子研究方法学。北京：人民卫生出版社。1999。
70 巴得年，何维。当代免疫学技术与应用。北京：北京医大中国协和医大联合出版社。1998。
71 Flynn JL, Goldstein MM, Triebold KJ, et al. Major histocompatibility complex class Ⅰ-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc. Natl. Acad. Sci. USA, 1992,89:12103.
72 Behar SM, Dascher CC, Grusby M J, et al. Susceptibility of mice deficient in CDID or TAP1 to infection with Mycobacterium tuberculosis. J. Exp. Med, 1999,189:1973.
73 Sousa AO, Mazzaccaro RJ, Russell RG, et al. Relative contributions of distinct MHC class Ⅰ-dependent cell populations in proteetin to tuberculosis infection in mice. Proc.Natl.Acad. Sci. USA,2000,97:4024.
74 Smith SM, Dockrell HM. Role of CD8 T cells in mycobacterial infections. Immunol Cell Biol, 2000,78(4):325-333.
75 Kamath AT, Groat NL, Bean AG, et al. Protective effect of DNA immunization against mycobacterial infection is associated with the early emergence of interferon-gamma (IFN-gamma)-secreting lymphocytes. Clin Exp Immunol. 2000,120(3):476-82.
76 Hernandez-Pando B, Orozece H, Sampieri A, et al. Correlation between the kinetics of Th1/Th2 and pathology in a murine modeel of experimental pulmonary tuberculosis. Immunol, 1996,89:26.
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7 Kaufman SH, Guile H, Daugelat S, et al. Tuberculosis and leprosy: attempts to identify T-cell antigens of potential value for vaccine design, Scand J Immunol Suppl; 1992,11:85-90．
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12 Vordermeier HM, Coombes AG, Jenkins P, et al. Synthetic delivery system for tuberculosis vaccines: immunological evaluation of the M. tuberculosis 38kDa protein entrapped in a biodegrdable PLG microparticles. Vaccine,1995,13:1576-1582．
13 Perales MA, Schwartz DH, Fabry JA, et al. A vaccinia-gp160-based vaccine but not gp160 protein vaccine elicits anti-gp160 cytotoxic T lymphocytes in some HIV-1 seronegative vaccinees. J Acquir Immune Defic Syndr Human Retrovirol,1995,10:27
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18 Aldovini A, Young RA. Humoral and cell-mediated immune responses to live recombinant BCG-HTV vaccines. Nature, 1992,351:479-482．
19 Connell ND, Medina-Acosta E, McMaster WR, et al. Effective immunization against cutaneous leishmaniasis with recombinant bacilli Calmette-Guerin expressing the Leishmania surface proteinase gp63． Proc. Natl.Acad.Sci.USA,1993,90:11473-11477．
20 Langermann S, Palaszynskl S, Sadiziene A, et al. Systemic and mucosal immunity induced by BCG vector expressing outer-surface protein A of Borrelia burgdorferi. Nature,1994,372:552-555．
21 Baumgart KW.Mckenzie KR, Radford AJ, et al. Immunogenicity and protection studies with recombinant mycobacteria and vaccinia vectors coexpressing the 18-kilodalton protein of Mycobacterium lepra. Infect Immun; 1996,64:2274-2281．
22 Behr MA, Wilon MA, Gill WP, et al. Comparative genomics of BCG vaccines by whole genome DNA microarry. Science, 1999;284:1520-1523．
23 Bardarov S, Kriakov J, Carriere C, et al. Development of conditional shuttle plasmid vectors of mycobacteriophages D29 and TM4: tools for transposon mutagenesis in mycobacteria(Abstract).96th General Meeting, American Society for Microbiology. P118．
24 Carol P. New method churus out TB mutants. Science, 1997,280:1607-1610．
25 Guleria I, Teitelbaum R, McAdam RA, et al. Auxotrophic vaccines for tuberculosis. Nat Med,1996,2:334-337．
26 Parish T, Stocker NG. Mycobacteria Protocols. Humana press, Totowa, New Jersey. 1998,p225-234．
27 De lisle GW, Wilson T, Collins DM, et al. Vaccination of guinea pigs with nutritionally impaired avirulent mutants of Mycobacterium bovis protects against tuberculosis. Infect Immun, 1999,67:2624-2626．
28 Jackson M, Phalen SW, Lagranderie M, et al. Persistence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine. Infect Immun, 1999,67:2867-2873．
29 Lowrie DB, Tascon RE, Colston MJ, et al. Towards a DNA vaccine against tuberculosis. Vaccine, 1994,12:1537-1540．
30 Lowrie DB, Silva CL, Colston MJ, et al. Protection against tuberculosis by a plasmid DNA vaccine.Vaccine,1997,15:834-838．
31 Bonato VL, Lima VM, Tascon RE, et al. Identification and characterization of protective T cells in hsp65 DNA-vaccined and M. tuberculosis-infected mice. Infect Immun, 1998,66:169-175．
32 Tacson RE,Colston MJ, Ragno S, et al. Vaccination against tuberculosis by DNA injection. Nat Med,1996,2:888-892．
33 Huygen K, Content J, Denis O, et al. Immunogenicity and protective
efficacy of a tuberculosis DNA vaccine. Nat Med, 1996,2:857-859．
34 Ulmer JB, Liu MA, Montgomery DL, et al. Expression and immunogenicity of M. tuberculosis antigen 85 by DNA vaccination. Vaccine,1997,15:792-794．
35 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. Vaccine, 1997,15:830-833．
36 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. Infect Immun, 1998,66:1527-1533．
37 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. Tuber Lung Dis, 1999,79:251-259．
38 Kamath AT, Feng CG, Macdonald M, et al. Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect Immun, 1999,67:1702-1707．
39 Li ZM, Howard A, Kelley C, et al. Immunogenicity of DNA vaccines expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences. Infect Immun,1999,67:4780-4786．
40 Morris S, Kelley C, Howard A, et al. The immunogenicity of single and combination DNA vaccines against tubeculosis. Vaccine,2000,18:2155-2163．
41 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. Infect Immun, 1999,67:2941-2950．
42 Zhu XJ, Venkataprasad N, Thangaraj HS, et al. Functins and specificity of T ells following nucleic acid vaccination of mice against Mycobacterium tuberculosis. J Immunol, 1997,158:5921-5926．
43 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. Infect Immun, 2000,68:1040-1047．
44 Erb KJ, Kirman J, Woodfield L, et al Identification of potential CD8+ T-cell epitopes of the 19kda and AhpC proteins from Mycobacterium tuberculosis. No evidence for CD8+T-cell priming against the identified peptides after DNA-vaccination of mice. Vaccine,1998,16:692-697．
45 Tanghe A, Lefevre P, Denis O, et al. Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors.J Immunol, 1999,162:1113-1119．
46 Chambers MA, Vordermeier H, Whelan A, et al. Vaccination of mice and cattle with plasmid DNA encoding the Mycobacterium bovis antigen MPB83． Clin Infect Dis,2000 ,30 (Suppl 3) :S283-287．
47 Tanghe A, Denis O, Lambrecht B, et al. Tuberculosis DNA vaccine encoding Ag85A is immunogenic and protective when administered by intramuscular needle injection but not by epidermal gene gun bombardment. Infect Immun, 2000,68(7) :3854-3860．
48 Kamath AT, Hanke T,Briscoe H, et al. Co-immunization with DNA vaccines expressing granulocyte-macrophage colony stimulating factor and mycobacterial secreted proteins enhances T-cell immunity, but not protective efficacy against Mycobacterium tuberculosis. Immunnol, 1999, 96: 511-516．
49 Chow YH, Chiang BL,Lee YL, et al. Development of Th1 and Th2 populations and the nature of immune responses to Hepatitis B virus DNA vaccines can be modulated by codelivery of various cytokine genes.J Immunol, 1998,160:1320-1329．
50 Tanghe A, D'Souza S, Rosseels V,et al. Improved immunogenicity and protective efficacy of a tuberculosis DNA vaccine encoding Ag85 by protein boosting. Infect Immun, 2001,69(5) :3041-3047．
51 Lowrie DB, Tascon RE, Bonato VLD, et al. Therapy of tuberculosis in mice by DNA vaccination. Nature, 1999,400:269-271．
52 Jungblut PR, Schaible UE, Mollenkopf HJ, et al. Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens. Mol-Microbiol, 1999,33: 1103-1117．
53 Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of
Mycobacterium tuberculosis from the complete genome sequence. Nature, 1998,393:537-544.
54 Walsh GP, et al. The Philippine cynomolgus monkey (Macaca fasicularis) provides a new nonhuman primate model of tuberculosis that resembles human disease. Nature Med. 1996,2:430-436.
55 Barry MA, Lai WC, Johnston SA. Protection against mycoplasma infection using expression-library immunization. Nature, 1995,377:632-635.
56 Sykes KF, Johnston SA. Linear expression elements: a rapid, in vivo, method to screen for gene functions. Nat Biotechnol, 1999,17(4):355-359.
57 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press, 1989.
58 Harth G, Lee BY, Horwitz MA. High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets. Infect Immun, 1997,65(6):2321-2328.
59 夏云，雷二庆，王槐春。Internet实用技术与生物医学应用。北京：军事医学科学出版社，1997。
60 孙志贤。现代生物化学理论与研究技术。北京：军事医学科学出版社，1995。
61 Tang DC, Devit M, Johnston SA, et al. Genetic immunization is a simple method for eliciting an immune response. Nature, 1992,356:152-154.
62 Tighe H, Corr M, Roman M, et al. Gene vaccination: plasmid DNA is more than just a blueprint. Immunol today, 1998,19(2):89-97.
63 Wiker HG, Harboe M. The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol Rev, 1992,56(4):648-661.
64 Torres M, Herrera T, Villareal H, et al. Cytokine profiles for peripheral blood lymphocytes from patients with active pulmonary tuberculosis and healthy household contacts in response to the 30-kilodalton antigen of Mycobacterium tuberculosis. Infect Immun, 1998,66(1):176-180.
65 Belisle JT, Vissa VD, Sievert T, et al. Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science 1997,276:1420-1422.
66 Silver RF, Wallis RS, Ellner JJ. Mapping of T cell epitopes of the 30-kDa antigen of Mycobacterium boris Bacillus Calmette-Guerin in purified protein derivative(PPD)- positve individuals. J Immunol., 1995,154:4665-4674.
67 Geluk A, van Meijgaarden KE, Franken KL, et al. Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A~*0201-restricted CD8+ T cells in HLA-transgenic mice and humans. J Immunol, 2000,165(11):6463-6471.
68 李国利，庄玉辉。分枝杆菌。北京：人民军医出版社。1985。
69 孙卫民，王惠琴。细胞因子研究方法学。北京：人民卫生出版社。1999。
70 巴得年，何维。当代免疫学技术与应用。北京：北京医大中国协和医大联合出版社。1998。
71 Flynn JL, Goldstein MM, Triebold KJ, et al. Major histocompatibility complex class Ⅰ-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc. Natl. Acad. Sci. USA, 1992,89:12103.
72 Behar SM, Dascher CC, Grusby M J, et al. Susceptibility of mice deficient in CDID or TAP1 to infection with Mycobacterium tuberculosis. J. Exp. Med, 1999,189:1973.
73 Sousa AO, Mazzaccaro RJ, Russell RG, et al. Relative contributions of distinct MHC class Ⅰ-dependent cell populations in proteetin to tuberculosis infection in mice. Proc.Natl.Acad. Sci. USA,2000,97:4024.
74 Smith SM, Dockrell HM. Role of CD8 T cells in mycobacterial infections. Immunol Cell Biol, 2000,78(4):325-333.
75 Kamath AT, Groat NL, Bean AG, et al. Protective effect of DNA immunization against mycobacterial infection is associated with the early emergence of interferon-gamma (IFN-gamma)-secreting lymphocytes. Clin Exp Immunol. 2000,120(3):476-82.
76 Hernandez-Pando B, Orozece H, Sampieri A, et al. Correlation between the kinetics of Th1/Th2 and pathology in a murine modeel of experimental pulmonary tuberculosis. Immunol, 1996,89:26.