结核分枝1402杆菌Rv3407基因的克隆与原核表达
摘要
结核病(Tuberculosis,TB)为全球性传染病,呈逐年增高趋势,给人类带来极大的危害,人们称之为白色瘟疫。近年来,由于卡介苗(Bacillus Caimette Guerin,BCG)保护性不完善、耐药结核分枝杆菌(Mycobacterium tuberculosis,MTB)的流行、与人类免疫缺陷病毒(Hunan immunodeficiency virus,HIV)的协同感染以及人口流动等原因使结核病疫情再度出现新高。目前全球有超过20亿人感染过结核分枝杆菌,每年新发TB病例超过800万,同时每年约有200～300万人死于TB,TB已成为世界上最严重的公共卫生问题之一。目前有效控制TB蔓延的主要策略有两方面:(1)活动性TB的快速诊断和及时治疗;(2)使用有效的疫苗阻止疾病的发生。因此进行TB新型疫苗或增强BCG免疫效果以及特异、快速诊断试剂的研究具有重要意义。
近十年来,人们对结核杆菌培养滤液中的蛋白进行了广泛的研究,并取得了令人鼓舞的结果。结核菌生长期间分泌的蛋白对结核病的保护性免疫是很重要的。国内外研究资料表明,结核分枝杆菌Rv3407基因含有300个碱基对的开放阅读框架(Open reading frame,ORF),该ORF编码99个氨基酸,编码蛋白分子质量为10kDa,结核分枝杆菌Rv3407蛋白是高度保守的有保守序列的假定蛋白(Conserved hypothetical protein),是结核杆菌生长期分泌的蛋白,具有细胞和体液免疫活性,国内还未见其结构和功能方面的研究,对其进行相关研究,可为结核疫苗的研制提供一定的实验依据。
本研究在国内外学者研究的基础上,运用现代分子生物学技术克隆了结核分枝杆菌Rv3407基因序列,并在大肠杆菌中得到表达,目的在于研究结核分枝杆菌Rv3407蛋白基因的结构和功能,为研制有效的亚单位疫苗奠定基础。在本研究中,以结核分枝杆菌H_(37)Rv的染色体DNA为摸板,PCR扩增了Rv3407蛋白基因,选用pGEM-Tvector system成功克隆了分枝杆菌Rv3407蛋白基因,经酶切鉴定和PCR鉴定,证实了所克隆的基因与目的基因大小一致。通过序列测定及DNASTAR分析发现,克隆的Rv3407蛋白基因序列与Genbank上发表的结核分枝杆菌H_(37)Rv菌株Rv3407蛋白基因序列同源性为100%,氨基酸序列也完全一致,表明该基因在结核分枝杆菌中是高度保守的。进一步将克隆至pGEM-T中的Rv3407蛋白基因亚克隆至pGEX-4T-1表达系统中,构建了高效原核表达质粒,在与之相匹配的E.coliBL21(DE 3)中经IPTG诱导4h后,表达量达到了高峰。本研究结果将进一步研究结核分枝杆菌基因工程亚单位疫苗及核酸疫苗莫定一定的基础。
Tuberculosis(TB)is a chronic respiratory infectious disease caused by pathogen Mycobacterium tuberculosis(MTB).In recent years,factors such as the unsuccessful protection of BCG,increasing frequency of drug-resistance MTB,incidence of HIV-associated tuberculosis and increasement of population movement,have aggravated further the opportunity of people worldwide face to the threat of TB.Currently. MTB has infected approximately two billion individuals in total.TB remains one of the great killers,causing between 2 and 3 million deaths,and an estimated more than 8 million new infections a year.There are two main strategies for reducing the transmission rate:(1)rapid diagnosis and promptly treatment of active TB;(2)preventing TB occurrence with an effective vaccine.There fore,it is very important to develop new vaccine,or enhance BCG effection and diagnosis reagent for finding TB patient timely and specially.
For the past ten years,people's has conducted the extensive research to bacillus tuberculosis nutrient fluid proteinand has obtained the inspiring result.The tubercidin bacillus growth period secretion's protein to tuberculosis's protection immunity is very important.The domestic and foreign research material indicated that the tubercidin bacillus Rv3407 protein is highly conservative has the conservative sequence hypothesis protein(Conserved hypothetical protein),is the bacillus tuberculosis vegetal period secretion protein,(99 aa) is composed of 300 bp,the code molecular is 10kDa,has the cell and humoral immunity activeness,domestic has not seen its structure and the function aspect research,research to it,may provide certain experiment basis for the tuberculosis vaccine development.
This research aims to study foreign scholars on the basis of the introduction of modern molecular biology techniques to Mycobacterium Determination of Rv3407 gene sequence and expression in Escherichia coli as the main research objective is to study the molecular people Mycobacterium biological characteristics, In order to develop an effective set of DNA-based vaccines.In this study,Mycobacterium tuberculosis Rv3407 gene was amplified from M.tuberculosis H_(37)Rv genomic DNA using PCR and use pGEM-T vector system successfully cloned human Mycobacterium Rv3407 protein genes,the identification and digested PCR,confirmed the the cloned gene consistent with the size of the target gene.Sequencing and DNASTAR through analysis found that the cloned Rv3407 protein gene sequence with Genbank publishes of Human Mycobacterium H_(37)Rv strain Rv3407 protein gene sequence homology of 100 percent,exactly the same amino acid sequence also showed that the gene in Mycobacterium is highly conserved.Further cloned in the pGEM-T-Rv3407 protein gene was subcloned into pGEX-4T-1 expression system to construct a highly efficient prokaryotic expression plasmid,in which the expression plasmid match E.coliBL21(DE 3)by IPTG induction 4 h,the expression level reached a peak,so as to further research to it,human genetic engineering Mycobacterium subunit vaccine and DNA vaccine for a certain foundation.
近十年来,人们对结核杆菌培养滤液中的蛋白进行了广泛的研究,并取得了令人鼓舞的结果。结核菌生长期间分泌的蛋白对结核病的保护性免疫是很重要的。国内外研究资料表明,结核分枝杆菌Rv3407基因含有300个碱基对的开放阅读框架(Open reading frame,ORF),该ORF编码99个氨基酸,编码蛋白分子质量为10kDa,结核分枝杆菌Rv3407蛋白是高度保守的有保守序列的假定蛋白(Conserved hypothetical protein),是结核杆菌生长期分泌的蛋白,具有细胞和体液免疫活性,国内还未见其结构和功能方面的研究,对其进行相关研究,可为结核疫苗的研制提供一定的实验依据。
本研究在国内外学者研究的基础上,运用现代分子生物学技术克隆了结核分枝杆菌Rv3407基因序列,并在大肠杆菌中得到表达,目的在于研究结核分枝杆菌Rv3407蛋白基因的结构和功能,为研制有效的亚单位疫苗奠定基础。在本研究中,以结核分枝杆菌H_(37)Rv的染色体DNA为摸板,PCR扩增了Rv3407蛋白基因,选用pGEM-Tvector system成功克隆了分枝杆菌Rv3407蛋白基因,经酶切鉴定和PCR鉴定,证实了所克隆的基因与目的基因大小一致。通过序列测定及DNASTAR分析发现,克隆的Rv3407蛋白基因序列与Genbank上发表的结核分枝杆菌H_(37)Rv菌株Rv3407蛋白基因序列同源性为100%,氨基酸序列也完全一致,表明该基因在结核分枝杆菌中是高度保守的。进一步将克隆至pGEM-T中的Rv3407蛋白基因亚克隆至pGEX-4T-1表达系统中,构建了高效原核表达质粒,在与之相匹配的E.coliBL21(DE 3)中经IPTG诱导4h后,表达量达到了高峰。本研究结果将进一步研究结核分枝杆菌基因工程亚单位疫苗及核酸疫苗莫定一定的基础。
Tuberculosis(TB)is a chronic respiratory infectious disease caused by pathogen Mycobacterium tuberculosis(MTB).In recent years,factors such as the unsuccessful protection of BCG,increasing frequency of drug-resistance MTB,incidence of HIV-associated tuberculosis and increasement of population movement,have aggravated further the opportunity of people worldwide face to the threat of TB.Currently. MTB has infected approximately two billion individuals in total.TB remains one of the great killers,causing between 2 and 3 million deaths,and an estimated more than 8 million new infections a year.There are two main strategies for reducing the transmission rate:(1)rapid diagnosis and promptly treatment of active TB;(2)preventing TB occurrence with an effective vaccine.There fore,it is very important to develop new vaccine,or enhance BCG effection and diagnosis reagent for finding TB patient timely and specially.
For the past ten years,people's has conducted the extensive research to bacillus tuberculosis nutrient fluid proteinand has obtained the inspiring result.The tubercidin bacillus growth period secretion's protein to tuberculosis's protection immunity is very important.The domestic and foreign research material indicated that the tubercidin bacillus Rv3407 protein is highly conservative has the conservative sequence hypothesis protein(Conserved hypothetical protein),is the bacillus tuberculosis vegetal period secretion protein,(99 aa) is composed of 300 bp,the code molecular is 10kDa,has the cell and humoral immunity activeness,domestic has not seen its structure and the function aspect research,research to it,may provide certain experiment basis for the tuberculosis vaccine development.
This research aims to study foreign scholars on the basis of the introduction of modern molecular biology techniques to Mycobacterium Determination of Rv3407 gene sequence and expression in Escherichia coli as the main research objective is to study the molecular people Mycobacterium biological characteristics, In order to develop an effective set of DNA-based vaccines.In this study,Mycobacterium tuberculosis Rv3407 gene was amplified from M.tuberculosis H_(37)Rv genomic DNA using PCR and use pGEM-T vector system successfully cloned human Mycobacterium Rv3407 protein genes,the identification and digested PCR,confirmed the the cloned gene consistent with the size of the target gene.Sequencing and DNASTAR through analysis found that the cloned Rv3407 protein gene sequence with Genbank publishes of Human Mycobacterium H_(37)Rv strain Rv3407 protein gene sequence homology of 100 percent,exactly the same amino acid sequence also showed that the gene in Mycobacterium is highly conserved.Further cloned in the pGEM-T-Rv3407 protein gene was subcloned into pGEX-4T-1 expression system to construct a highly efficient prokaryotic expression plasmid,in which the expression plasmid match E.coliBL21(DE 3)by IPTG induction 4 h,the expression level reached a peak,so as to further research to it,human genetic engineering Mycobacterium subunit vaccine and DNA vaccine for a certain foundation.
引文
[1]Dye C,Scheele S,Dolin P,et al.Consensus statement.Global burden of tuberculosis:estimated incidence,prevalence and mortality by country.WHO Global Surveillance and Monitoring Project.[J].AMA,1999,282(7):677-686
[2]Wiker HG,Mustafa T,Malen H,et al.Vaccine approaches to prevent tuberculosis.[J].Scand J Immunol,2006,64(3):243-250
[3]Colditz GA,Brewer TF,Berkey CS,et al.Efficacy of BCG vaccine in the prevention of tuberculosis.Meta-analysis of the published literature.[J].JAMA,1994,271(9):698-702
[4]Fine PE.Variation in protection by BCG:implications of and for heterologous immunity.[J].Lancet,1995,18;346(8986):1339-45.Review.Erratum in:Lancet1996,347(8997):340
[5]Andersen P,Doherty TM.The success and failure of BCG-implicadons for a novel tuberculosis vaccine.Nat Rev Microbiol,2005,3(8):656-662
[6]Haile M,Kallenius G.Recent developments in tuberculosis vaccines.Curr Opin Infect Dis,2005,18(3):211-215
[7]Kaufmann SH.Introduction.Rational vaccine development against tuberculosis:‘Those who don't remember the past are condemned to repeat it'.Microbes Infect,2005,7(5-6):897-898
[8]Kaufmarm SH.Envisioning future strategies for vaccination against tuberculosis.Nat Rev Immunol,2006,6(9):699-704
[9]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 spectrometry.Infect.Immun,1997,65:4515-4524
[10]AndersenP,Andersen AB,Sorensen AL,Nagai S.Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice.[J]Immunol,1995,154:3359-3372
[11]张立兴,屠德华,阐冠卿.国际结核病流行出现的严重问题和我们的对策.[J].中国防痨杂志,1996,19:97-100
[12]Nunn P.The Qlobal Epidemic the Present Epidemiology of Tuberculosis.[J].Scot Med J,2000,45(1)6-7
[13]姜世闻,徐汉成,李继海.高校保健医学研究与实践.[J].2006(1)
[14]Mustafa Abu S,AI-Attiyah R.Tuberculosis:looking beyond BCG vaccine.J Postgrad Med.2003,49:134-140
[15]何昭阳.牛结核病研究进展.预防兽医学研究进展,2001,3(4):34-39
[16]于春光,姚贵芳.鹿结核病的诊治.[J].黑龙江畜牧兽医,2004,4:36
[17]刘桂香,邵锡如,田登,等.应用DNA基因诊断白唇鹿结核杆菌的感染.[J].医学动物防制,2002,18(6):293-294
[18]Griffin J FT,Buchan GS.Aetiology pathogenesis and diagnosis of Mycobacterium bovis in deer. [J].Veterinary Microbiology,1994,40:193-205
[19]丁春光,姚贵芳.鹿结核病的诊治.[J].黑龙江畜牧兽医,2004,(4):36
[20]丁润峰,戴春玲,丁世哲,等.关于鹿结核病及其防制措施的探讨.[J].当代畜牧,2004(3):18-20
[21]李太元,金吉东,于文会,等.鹿结核病的诊断、病原分离及鉴定.[J].黑龙江畜牧兽医,2002(11):34-35
[22]张旭静.小种群白唇鹿结核病的流行与诊断.[J].中国预防兽医学报,2002,24(2):23-24
[23]刘桂香,邵锡如,等.应用DNA基因诊断白唇鹿结核杆菌的感染.[J].医学动物防制,2002,18(6):293-294
[24]蔡宝祥.家畜传染病学.中国农业出版社,2001,110-114
[25]王春芳.牛分枝杆菌MPB64基因的克隆与原核表达.[吉林农业大学优秀硕士论文].吉林农业大学,2005
[26]Aderem A,Underhill DM.Mechanisms of phagocytosis in macrophage.Annu Rev Immunol.1999,17:592-623
[27]Schorey JS,Carroll MC,Brown FJ.A macrophage invasion mechanism of mycobacteria.Science,1999,277:1091-1093
[28]Steffen Stenger,Robert L,Modlin.T cell mediated immunity to Mycobacterium tuberculosis.Current Opinion in Microbiology,1999,2:89-93
[29]Havlir DV,Ellner JJ,Chervenak KA,et al.Selective expansion of human gamma delta T cells by monocytes infected with live Mycobacterium tuberculosis.J Clin Invest.,1991,87(2):729-733
[30]Bukowski JF,Morita CT,Tanaka Y,et al.V ganmma 2V delta 2 TCR-dependent recognition of non-peptide antigens and Daudi cells analyzed by TCR gene transfer.[J].Immunol.1995,154(3):998-1006
[31]Cole ST,Brosch R,Parkhill J,et al.Deciphering the biology of Mycobaeterium tuberculosis from the complete genome sequence.Nature,1998,393:537-544
[32]Morris S,Kelley C,Howard A,et al.The immunogenicity of single and combination DNA vaccines against tuberculosis.Vaccine,2000,18:2155-2163
[33]Brjker M.New approaches to vaccines against tuberculosis:where we stand and where we want to go?Immunol Microbiol,1999,23:147-148
[34]Orme IM.Progress in the development of new vaccines against tuberculosis.Int J Tuberc Lung Dis,1997,1(2):95-100
[35]Ginsberg A M.What's new in tuberculosis vaccine? Bull WHO,2002 80(6):423-438
[36]吴悦涵.结核分枝杆菌的分子免疫学进展.国外医学免疫学,2004,27(2):75-80
[37]Jackson M,Phalen SW.Persistence and protective efficacy of a Mycobacterium tuberculosis Auxotroph Vaccine.Infect & Immun,1999,67(6):2867-2873
[38]Jackson M,Phalen S W,Lagranderie M,et al.Persistence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine.Infect Immun,1999,67:2867-2873
[39]Martin S,Pavelka Jr,William R,Jacobs Jr.Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis,Mycobacterium bovis Bacillus Calmette-Gu()rin and Myeobacterium tuberculosis H_(37)Rv by Allelic Exchange.J Bacerio,1999,181(16):4780-4789
[40]Jungblut PR,Schaible U E.Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains:towards functional genomics of microbial pathogens.Mol Microbiol,1999,33(6):1103-1117
[41]Geluk A,Taneja V.Identification of HLA class Ⅱ-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic,class Ⅱ-deficient mice.PNAS,1998,95(18):10797-10802
[42]Cockle P J,Gordon SV,Lalvani A,et al.Identification of novel Mycobacterium tuberculosis antigens with potential as diagnostic reagents or subunit vaccine candidates by comparative genomics.Infect & Immun,2002,70(12):6996-7003
[43]Lakey DL,Voladri RK,Edwards KM,et al.Enhanced production of recombinant Mycobacterium tuberculosis antigens in Escherichia coli by replacement of low-usage codons.Infect & Immun,2000,68(1):233-238
[44]Erb KJ,Kirman J,Woodfield L,et al.Indentification 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,1998,1:692-697
[45]Wolff JA,Malone RW,Williams P,et al.Direct gene transfer into mouse musclein vivo.Science,1990,247:1465-1468
[46]Tang DC,DeVit M,Johnston SA.Genetic immunization is a simple method for eliciting an immune response.Nature,1992,356:152-154
[47]Leitner WW,Ying H,Restifo NP.DNA and RNA-based vaccines:principle,progress and prospects.Vaccine,1999,18:765-767
[48]王宝林,王洪海.结核分枝杆菌DNA疫苗研究进展.中华结核和呼吸杂志,2004,27(11):787-790
[49]Taubes G.Immunology:Ways to Vary the Gene Vaccine Theme.Science,1997,278(5344):1713
[50]Goonetilleke NP,McShane H,Hannan CM,et al.Enhanced immunogenicity and protective efficacy against Mycobacterium tuberculosis of Bacille Calmette Gu(?)in vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus Ankara.Immunol,2003,171(3):1602-1609
[51]Triccas JA,Sun L,Palendira U,et al.Comparative affects of plasmid encoded Interleukin 12 and interleukin18 on the protective efficacy of DNA vaccination against Mycobacterium tuberculosis.Immunol Cell Biol,2002,80(4):346-350
[52]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 vaccines.[J] Acquir Immune Defic Syndr Human Retrovirol,1995,10-27
[53] Borysiewicz LK, Fiander A, Nimako M, et al.A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer. Lancet,1996,347:1523
[54] Zhu X, Venkataprasad N, Ivanyi J, et al.Vaccination with recombinant vaccinia virus protects mice against Mycobacterium tuberculosis infection. Immunology, 1997,92:6-9
[55] LyonsJ, Sinos C, Destree A, et al Expression of Mycobacterium tuberculosis and Mycobacterium proteins by vaccinia virus.Infect Immun,1990,58:4089-4098
[56] Hess J, Grode L, Hellwig J, et al. Protection against marine tuberculosis by an recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG. FEMS Immunol Med Microbiol,2000,27(4):283-289
[57] Wade MM,Zhang Y .Mechanisms of drug resistance in Mycobacterium tuberculosis.[J].Front Biosci,2004, 9:975-994
[58] Zhang Y, Heym B, Allen B, et al.The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis.[J]. Nature, 1992 358(6387):591-593
[59] Zhang Y, Garbe T, Young D. Transformation with katG restores isoniazid-sensitivity in Mycohacterium tuberculosis isolates resistant to a range of drug concentrations. Mol Microbiol.1993, 8(3): 521-524
[60] Wilson TM, Clollins DM. ahpC, a gene involved in isoniazid resistanceof the Mycobacterium tuberculosis complex. Mol Microbiol.1996,19:1025-1034
[61] Yue J, ShiW,Xie J, et al. Mutations in the rpoB gene ofmultidrug-resistant Mycobacterium tuberculosis isolates from China [J]. J Clin Microbiol, 2003, 41(5):2209-2212
[62] Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agentresistance in Mycobacterium tuberculosis: 1998 update [J].Tuber Lung Dis,1998,79(1):3-29
[63] Davies J, Davis BD. Misreading of ribonucleic acid code words induced by aminoglycoside antibiotics.The effect of drug concentration.[J].J Biol Chem.1968, 243(12):3312-3316
[64] Douglass J, Steyn LM. A ribosomal gene mutation in streptomycin-resistant Mycobacterium tuberculosis isolates.[J].J Infect Dis, 1993, 167(6):1505-1506
[65] Finken M, Kirschner P, Meier A, et al. Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of theribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot.[J].Mol Microbiol, 1993, 9(6): 1239-1246
[66] Srecvatson S, Pan X, Stockbauer KE, et al. Ethambutol resistance in Mycobacterium tubercrulosis:critical role of embB mutations. Antimicrob Agents Chemother.1996,40:1024-1026
[67] Honore N, Cole ST. Implications of short-course chemotherapy of multidrug resistance for the future of tuberculosis:a molecular study.Antimierob Agents Chemother,1994,38(2):238-242
[68]Cooksey RC,Morlock GP,McQueen A,et al.Characterization of streptomycin resistance mechanisms among Mycobacterium tuberculosis isolates from patients in New York City.[J].Antimicrob Agents Chemother,1996,40(5):1186-1188
[69]Takayama K,Kilburn JO.Inhibition of synthesis of arabinogalactan by ethambutol in Mycobacterium smegmatis.[J].Antimicrob Agents Chemother,1989,33(9):1493-1499
[70]Belanger AE,Besra GS,Ford ME,et al.The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol.[J].Proc Natl Acad Sci U S A,1996,93(21):11919-11924
[71]Scorpio A,Zhang Y Mutations in pncA.a gene encoding pyrazinamidase/nicotinamidase,cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus.[J].Nat Med,1996,2(6):662-667
[72]Zhang Y,Permar S,Sun Z.Conditions that may affect the results of susceptibility testing of Mycobacterium tuberculosis to pyrazinamide.[J].J Med Microbiol,2002,51(1):42-49
[73]Scorpio A,Lindholm-Levy P,Heifets L,et al.Characterization of pncA mutationsin pyrazinamide-resistant Mycobacterium tuberculosis.[J].Antimicrob AgentsChemother,1997,41(3):540-543
[74]Cheng S J,Thibert L,Sanchez T,et al.pncA mutations as a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis:spread of a monoresistant strain in Quebec,Canada.[J].Antimicrob Agents Chemother,2000,44(3):528-32
[75]Scorpio A,Zhang Y.Mutations in pncA,a gene encoding pyrazinamidase/nicotinamidase,cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus.Nat Med.1996,2(6):662-667
[76]Takiff HE,Salazar L,Guerrero C,et al.Cloning and nucleotidesequence of Mycobacterium tuberculosis gyrA and gyrB genes anddetection of quinolone resistance mutations.Antimicrob.Agents.Chemother.1994,38:773-780
[77]吴传勇,娄加陶,蒋廷旺,等.结核杆菌抗原多表位融合蛋白的重组表达与鉴定.[J].第二军医大学学报,2006,27(12):1281
[78]叶棋浓,苏国富.重组人GM-CSF/MCAF融合蛋白的变性、复性及纯化研究[J].生物工程学报,1996,4(12):434-439
[2]Wiker HG,Mustafa T,Malen H,et al.Vaccine approaches to prevent tuberculosis.[J].Scand J Immunol,2006,64(3):243-250
[3]Colditz GA,Brewer TF,Berkey CS,et al.Efficacy of BCG vaccine in the prevention of tuberculosis.Meta-analysis of the published literature.[J].JAMA,1994,271(9):698-702
[4]Fine PE.Variation in protection by BCG:implications of and for heterologous immunity.[J].Lancet,1995,18;346(8986):1339-45.Review.Erratum in:Lancet1996,347(8997):340
[5]Andersen P,Doherty TM.The success and failure of BCG-implicadons for a novel tuberculosis vaccine.Nat Rev Microbiol,2005,3(8):656-662
[6]Haile M,Kallenius G.Recent developments in tuberculosis vaccines.Curr Opin Infect Dis,2005,18(3):211-215
[7]Kaufmann SH.Introduction.Rational vaccine development against tuberculosis:‘Those who don't remember the past are condemned to repeat it'.Microbes Infect,2005,7(5-6):897-898
[8]Kaufmarm SH.Envisioning future strategies for vaccination against tuberculosis.Nat Rev Immunol,2006,6(9):699-704
[9]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 spectrometry.Infect.Immun,1997,65:4515-4524
[10]AndersenP,Andersen AB,Sorensen AL,Nagai S.Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice.[J]Immunol,1995,154:3359-3372
[11]张立兴,屠德华,阐冠卿.国际结核病流行出现的严重问题和我们的对策.[J].中国防痨杂志,1996,19:97-100
[12]Nunn P.The Qlobal Epidemic the Present Epidemiology of Tuberculosis.[J].Scot Med J,2000,45(1)6-7
[13]姜世闻,徐汉成,李继海.高校保健医学研究与实践.[J].2006(1)
[14]Mustafa Abu S,AI-Attiyah R.Tuberculosis:looking beyond BCG vaccine.J Postgrad Med.2003,49:134-140
[15]何昭阳.牛结核病研究进展.预防兽医学研究进展,2001,3(4):34-39
[16]于春光,姚贵芳.鹿结核病的诊治.[J].黑龙江畜牧兽医,2004,4:36
[17]刘桂香,邵锡如,田登,等.应用DNA基因诊断白唇鹿结核杆菌的感染.[J].医学动物防制,2002,18(6):293-294
[18]Griffin J FT,Buchan GS.Aetiology pathogenesis and diagnosis of Mycobacterium bovis in deer. [J].Veterinary Microbiology,1994,40:193-205
[19]丁春光,姚贵芳.鹿结核病的诊治.[J].黑龙江畜牧兽医,2004,(4):36
[20]丁润峰,戴春玲,丁世哲,等.关于鹿结核病及其防制措施的探讨.[J].当代畜牧,2004(3):18-20
[21]李太元,金吉东,于文会,等.鹿结核病的诊断、病原分离及鉴定.[J].黑龙江畜牧兽医,2002(11):34-35
[22]张旭静.小种群白唇鹿结核病的流行与诊断.[J].中国预防兽医学报,2002,24(2):23-24
[23]刘桂香,邵锡如,等.应用DNA基因诊断白唇鹿结核杆菌的感染.[J].医学动物防制,2002,18(6):293-294
[24]蔡宝祥.家畜传染病学.中国农业出版社,2001,110-114
[25]王春芳.牛分枝杆菌MPB64基因的克隆与原核表达.[吉林农业大学优秀硕士论文].吉林农业大学,2005
[26]Aderem A,Underhill DM.Mechanisms of phagocytosis in macrophage.Annu Rev Immunol.1999,17:592-623
[27]Schorey JS,Carroll MC,Brown FJ.A macrophage invasion mechanism of mycobacteria.Science,1999,277:1091-1093
[28]Steffen Stenger,Robert L,Modlin.T cell mediated immunity to Mycobacterium tuberculosis.Current Opinion in Microbiology,1999,2:89-93
[29]Havlir DV,Ellner JJ,Chervenak KA,et al.Selective expansion of human gamma delta T cells by monocytes infected with live Mycobacterium tuberculosis.J Clin Invest.,1991,87(2):729-733
[30]Bukowski JF,Morita CT,Tanaka Y,et al.V ganmma 2V delta 2 TCR-dependent recognition of non-peptide antigens and Daudi cells analyzed by TCR gene transfer.[J].Immunol.1995,154(3):998-1006
[31]Cole ST,Brosch R,Parkhill J,et al.Deciphering the biology of Mycobaeterium tuberculosis from the complete genome sequence.Nature,1998,393:537-544
[32]Morris S,Kelley C,Howard A,et al.The immunogenicity of single and combination DNA vaccines against tuberculosis.Vaccine,2000,18:2155-2163
[33]Brjker M.New approaches to vaccines against tuberculosis:where we stand and where we want to go?Immunol Microbiol,1999,23:147-148
[34]Orme IM.Progress in the development of new vaccines against tuberculosis.Int J Tuberc Lung Dis,1997,1(2):95-100
[35]Ginsberg A M.What's new in tuberculosis vaccine? Bull WHO,2002 80(6):423-438
[36]吴悦涵.结核分枝杆菌的分子免疫学进展.国外医学免疫学,2004,27(2):75-80
[37]Jackson M,Phalen SW.Persistence and protective efficacy of a Mycobacterium tuberculosis Auxotroph Vaccine.Infect & Immun,1999,67(6):2867-2873
[38]Jackson M,Phalen S W,Lagranderie M,et al.Persistence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine.Infect Immun,1999,67:2867-2873
[39]Martin S,Pavelka Jr,William R,Jacobs Jr.Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis,Mycobacterium bovis Bacillus Calmette-Gu()rin and Myeobacterium tuberculosis H_(37)Rv by Allelic Exchange.J Bacerio,1999,181(16):4780-4789
[40]Jungblut PR,Schaible U E.Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains:towards functional genomics of microbial pathogens.Mol Microbiol,1999,33(6):1103-1117
[41]Geluk A,Taneja V.Identification of HLA class Ⅱ-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic,class Ⅱ-deficient mice.PNAS,1998,95(18):10797-10802
[42]Cockle P J,Gordon SV,Lalvani A,et al.Identification of novel Mycobacterium tuberculosis antigens with potential as diagnostic reagents or subunit vaccine candidates by comparative genomics.Infect & Immun,2002,70(12):6996-7003
[43]Lakey DL,Voladri RK,Edwards KM,et al.Enhanced production of recombinant Mycobacterium tuberculosis antigens in Escherichia coli by replacement of low-usage codons.Infect & Immun,2000,68(1):233-238
[44]Erb KJ,Kirman J,Woodfield L,et al.Indentification 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,1998,1:692-697
[45]Wolff JA,Malone RW,Williams P,et al.Direct gene transfer into mouse musclein vivo.Science,1990,247:1465-1468
[46]Tang DC,DeVit M,Johnston SA.Genetic immunization is a simple method for eliciting an immune response.Nature,1992,356:152-154
[47]Leitner WW,Ying H,Restifo NP.DNA and RNA-based vaccines:principle,progress and prospects.Vaccine,1999,18:765-767
[48]王宝林,王洪海.结核分枝杆菌DNA疫苗研究进展.中华结核和呼吸杂志,2004,27(11):787-790
[49]Taubes G.Immunology:Ways to Vary the Gene Vaccine Theme.Science,1997,278(5344):1713
[50]Goonetilleke NP,McShane H,Hannan CM,et al.Enhanced immunogenicity and protective efficacy against Mycobacterium tuberculosis of Bacille Calmette Gu(?)in vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus Ankara.Immunol,2003,171(3):1602-1609
[51]Triccas JA,Sun L,Palendira U,et al.Comparative affects of plasmid encoded Interleukin 12 and interleukin18 on the protective efficacy of DNA vaccination against Mycobacterium tuberculosis.Immunol Cell Biol,2002,80(4):346-350
[52]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 vaccines.[J] Acquir Immune Defic Syndr Human Retrovirol,1995,10-27
[53] Borysiewicz LK, Fiander A, Nimako M, et al.A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer. Lancet,1996,347:1523
[54] Zhu X, Venkataprasad N, Ivanyi J, et al.Vaccination with recombinant vaccinia virus protects mice against Mycobacterium tuberculosis infection. Immunology, 1997,92:6-9
[55] LyonsJ, Sinos C, Destree A, et al Expression of Mycobacterium tuberculosis and Mycobacterium proteins by vaccinia virus.Infect Immun,1990,58:4089-4098
[56] Hess J, Grode L, Hellwig J, et al. Protection against marine tuberculosis by an recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG. FEMS Immunol Med Microbiol,2000,27(4):283-289
[57] Wade MM,Zhang Y .Mechanisms of drug resistance in Mycobacterium tuberculosis.[J].Front Biosci,2004, 9:975-994
[58] Zhang Y, Heym B, Allen B, et al.The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis.[J]. Nature, 1992 358(6387):591-593
[59] Zhang Y, Garbe T, Young D. Transformation with katG restores isoniazid-sensitivity in Mycohacterium tuberculosis isolates resistant to a range of drug concentrations. Mol Microbiol.1993, 8(3): 521-524
[60] Wilson TM, Clollins DM. ahpC, a gene involved in isoniazid resistanceof the Mycobacterium tuberculosis complex. Mol Microbiol.1996,19:1025-1034
[61] Yue J, ShiW,Xie J, et al. Mutations in the rpoB gene ofmultidrug-resistant Mycobacterium tuberculosis isolates from China [J]. J Clin Microbiol, 2003, 41(5):2209-2212
[62] Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agentresistance in Mycobacterium tuberculosis: 1998 update [J].Tuber Lung Dis,1998,79(1):3-29
[63] Davies J, Davis BD. Misreading of ribonucleic acid code words induced by aminoglycoside antibiotics.The effect of drug concentration.[J].J Biol Chem.1968, 243(12):3312-3316
[64] Douglass J, Steyn LM. A ribosomal gene mutation in streptomycin-resistant Mycobacterium tuberculosis isolates.[J].J Infect Dis, 1993, 167(6):1505-1506
[65] Finken M, Kirschner P, Meier A, et al. Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of theribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot.[J].Mol Microbiol, 1993, 9(6): 1239-1246
[66] Srecvatson S, Pan X, Stockbauer KE, et al. Ethambutol resistance in Mycobacterium tubercrulosis:critical role of embB mutations. Antimicrob Agents Chemother.1996,40:1024-1026
[67] Honore N, Cole ST. Implications of short-course chemotherapy of multidrug resistance for the future of tuberculosis:a molecular study.Antimierob Agents Chemother,1994,38(2):238-242
[68]Cooksey RC,Morlock GP,McQueen A,et al.Characterization of streptomycin resistance mechanisms among Mycobacterium tuberculosis isolates from patients in New York City.[J].Antimicrob Agents Chemother,1996,40(5):1186-1188
[69]Takayama K,Kilburn JO.Inhibition of synthesis of arabinogalactan by ethambutol in Mycobacterium smegmatis.[J].Antimicrob Agents Chemother,1989,33(9):1493-1499
[70]Belanger AE,Besra GS,Ford ME,et al.The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol.[J].Proc Natl Acad Sci U S A,1996,93(21):11919-11924
[71]Scorpio A,Zhang Y Mutations in pncA.a gene encoding pyrazinamidase/nicotinamidase,cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus.[J].Nat Med,1996,2(6):662-667
[72]Zhang Y,Permar S,Sun Z.Conditions that may affect the results of susceptibility testing of Mycobacterium tuberculosis to pyrazinamide.[J].J Med Microbiol,2002,51(1):42-49
[73]Scorpio A,Lindholm-Levy P,Heifets L,et al.Characterization of pncA mutationsin pyrazinamide-resistant Mycobacterium tuberculosis.[J].Antimicrob AgentsChemother,1997,41(3):540-543
[74]Cheng S J,Thibert L,Sanchez T,et al.pncA mutations as a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis:spread of a monoresistant strain in Quebec,Canada.[J].Antimicrob Agents Chemother,2000,44(3):528-32
[75]Scorpio A,Zhang Y.Mutations in pncA,a gene encoding pyrazinamidase/nicotinamidase,cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus.Nat Med.1996,2(6):662-667
[76]Takiff HE,Salazar L,Guerrero C,et al.Cloning and nucleotidesequence of Mycobacterium tuberculosis gyrA and gyrB genes anddetection of quinolone resistance mutations.Antimicrob.Agents.Chemother.1994,38:773-780
[77]吴传勇,娄加陶,蒋廷旺,等.结核杆菌抗原多表位融合蛋白的重组表达与鉴定.[J].第二军医大学学报,2006,27(12):1281
[78]叶棋浓,苏国富.重组人GM-CSF/MCAF融合蛋白的变性、复性及纯化研究[J].生物工程学报,1996,4(12):434-439