沙眼衣原体J型野生株ompA基因重组子的构建
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摘要
衣原体是专性细胞内寄生的原核微生物，革兰氏染色阴性，属细菌范畴。衣原体引起的疾病相当广泛：在发达国家衣原体是性传播疾病的首位病原体，由它引起的非淋菌性尿道炎的发病率超过淋病，在性传播疾病中己跃居首位；沙眼衣原体(C．t)感染眼部引起的沙眼曾肆虐全世界，至今仍是全球可预防性眼盲的首要原因；衣原体感染动物可导致牛、羊流产，母鸡不能正常产卵，对养殖畜牧业造成很大危害；因此衣原体越来越成为人们关注的重要病原体。人们急切地期待衣原体疫苗的出现以控制衣原体的感染及蔓延，科技工作者在此领域已探索50余年，曾试验过多种疫苗，如：灭活或减毒的衣原体疫苗、亚单位疫苗、树突状细胞疫苗等，但迄今尚无成熟疫苗问世。近年来DNA疫苗研究的快速发展给衣原体疫苗研制带来新的希望，随着分子生物学技术突飞猛进的发展，基因工程技术日臻完善，衣原体的生物构成及基因序列日益明朗，衣原体的DNA疫苗很有发展潜力。
DNA疫苗是运用基因工程技术，将编码某种蛋白的外源基因与细菌质粒构建的重组体直接免疫机体，转染宿主细胞，使其表达保护性抗原，从而诱导机体产生特异性免疫的疫苗。
本实验的目的是构建C．t主要外膜蛋白(MOMP)编码基因ompA与pcDNA3质粒的重组体ompA-pcDNA3，即C．t的DNA疫苗，希望能为C．tDNA疫苗的动物实验研究提供材料。
实验中选用的真核表达载体为pcDNA3质粒，目的基因为C．t的MOMP编码基因ompA，使用的C．t标本来源于性传播疾病门诊患者的尿道及宫颈分泌物。标本经裂解处理后做为PCR扩增的模板，先用C．t种特异性引物扩增ompA基因，以此来筛选C．t阳性的临床标本；PCR产物经基因测序，确定C．t临床标本的血清型；然后用带限制性酶切位点的血清型特异性引物，PCR扩增此特定血清型的ompA基因；扩增的目的基因与载体经酶切后，连接重组，转化大肠杆菌；最后重组子经抗生素平板筛选、PCR扩增筛选、酶切鉴定后，阳性重组子送测序公司，进行重组子的DNA序列测定。
实验显示：PCR扩增方法筛选C．t阳性的临床标本，扩增产物经琼脂糖凝胶电泳，扩增产物大小约1.2Kb，基因测序确定是C．t J血清型；PCR扩增
天津医科大学硕士研究生论文
J型c.t MoMP编码基因。mPA，扩增产物经琼脂糖凝胶电泳，扩增产物片段
约1.OKb，与预期的1022bp一致;PCR扩增方法筛选重组子ompA一PcDNA3，
琼脂糖凝胶电泳提示。mpA基因存在于PcDNA3质粒中;双酶切法筛选重组
子，酶切产生了5.4Kb和1 .oKb两个片段，进一步证明omPA基因存在于
PcDNA3质粒中;阳性重组子进行基因测序，证明插入PcDNA3载体中的墓
因片段与genebar水中J型C .t的 omPA基因序列一致。上述结果证明本实验
成功构建了C.tJ型野生株。mpA基因的重组子ompA一peDNA3。
Chlamydia is an obligate , intracellular, Gram negative bacterial pathogen. The diseases evoked by chlamydia are quite wide. In developed countries, the pathogen is the leading cause of sexually transmitted disense. Infection in the eyes results in trachoma which once spreaded widely in the whole world. By now trachoma is still the leading cause of preventable blindness in the world. Chlamydia infection can also make cattle and sheep abort, hens not lay eggs normally. So chlamydia has made huge damage to both mankind and anminals. Chlamydia has became the important pathogen which absorbs people's attention. Chlamydial vaccion has the probability of controling chlamydial infection and spread. People have studied chlamydia vaccine for more then 50 years. Many kinds of vaccine have been tested, such as the inactivated pathogen vaccine, the live attenuated pathogen vaccine, the subunit vaccine, the dendritic cell vaccine and so on. But there isa't a mature vaccine that can be used now. In recent years, the fast
and deep research OB the DNA vaccine brings new hope for stydying the chlamydia DNA vaccine.
The DNA vaccine, which is a recombinant of plasmid with a gene fagment encoding antigen protein, vaccinate the organism directly , transform the organism cells, express the protective antigen, and then induce the organism to obtain specefic immunity.
Our test is aming at constructing an recombinant of ompA gene from the chlamydia trachomatis wild strain. We choose pcDNA3 plasmid as the vector and ompA gene which encodes chlamydia trachomatis (C.t) major outer membrane protein as the target DNA. Clinical chlamydia trachomatis samples,urethral and cervical secreations, were collected from persons who presented to the outpatient department of the sexually transmitted disease. After lysised, clinical samples wert used as template for polymerase chain reaction(PCR). Firstly ,to select C.t positive
samples, we amplified ompA gene through PCR using C.t species specific primier, and then we ascertain the C.t clinical specimen's serotype by the DNA sequence method. Secondly, through polymerase chain reaction, we amplified specific serotype C.t ompA gene using serotype specific primier. Then we enzymatically inserted DNA fragment into plasmid vector, we used three methods to screen positive recombinant, including culturing the E.coli on LB agarose plate containing ampicillin, amplifing ompA gene from recomibant through the PCR method, and digesting recombinant by restriction endonuclease. Finally, we sequenced the inserted DNA fragment by sending the positive recombinant to the sequence corporation.
The results are as follows. By using the PCR method to screen positive C.t clinical samples, the obtained DNA fragment is about 1.2Kb long. The sample which was sended to be sequenced was proved to be C.t J serotype. Agarose gel electrophoresis manifests that the amplified C.t J serotype ompA gene is a little longer than 1.0Kb. The length of the amplified DNA fragment is consistent whih what we have anticipated. When positive recombinant is screened by the PCR method, agarose gel electrophoresis manifests that ompA gene lies in pcDNA3 plasmid. Digested recombinant with restriction endonuclease, we obtained two DNA fragments of separately 5.4Kb and 1.0Kb. The result also confirms that the ompA gene lies in pcDNA3 plasmid. When we sequenced the inserted DNA fragment, the result proved that the inserted gene has the same sequence with C.t J serotype ompA gene provided by genebank. Above results manifest that our test have successfully construsted an recombinant of ompA gene from chlamydia trachomatis wild strai
n J serotype.
DNA疫苗是运用基因工程技术，将编码某种蛋白的外源基因与细菌质粒构建的重组体直接免疫机体，转染宿主细胞，使其表达保护性抗原，从而诱导机体产生特异性免疫的疫苗。
本实验的目的是构建C．t主要外膜蛋白(MOMP)编码基因ompA与pcDNA3质粒的重组体ompA-pcDNA3，即C．t的DNA疫苗，希望能为C．tDNA疫苗的动物实验研究提供材料。
实验中选用的真核表达载体为pcDNA3质粒，目的基因为C．t的MOMP编码基因ompA，使用的C．t标本来源于性传播疾病门诊患者的尿道及宫颈分泌物。标本经裂解处理后做为PCR扩增的模板，先用C．t种特异性引物扩增ompA基因，以此来筛选C．t阳性的临床标本；PCR产物经基因测序，确定C．t临床标本的血清型；然后用带限制性酶切位点的血清型特异性引物，PCR扩增此特定血清型的ompA基因；扩增的目的基因与载体经酶切后，连接重组，转化大肠杆菌；最后重组子经抗生素平板筛选、PCR扩增筛选、酶切鉴定后，阳性重组子送测序公司，进行重组子的DNA序列测定。
实验显示：PCR扩增方法筛选C．t阳性的临床标本，扩增产物经琼脂糖凝胶电泳，扩增产物大小约1.2Kb，基因测序确定是C．t J血清型；PCR扩增
天津医科大学硕士研究生论文
J型c.t MoMP编码基因。mPA，扩增产物经琼脂糖凝胶电泳，扩增产物片段
约1.OKb，与预期的1022bp一致;PCR扩增方法筛选重组子ompA一PcDNA3，
琼脂糖凝胶电泳提示。mpA基因存在于PcDNA3质粒中;双酶切法筛选重组
子，酶切产生了5.4Kb和1 .oKb两个片段，进一步证明omPA基因存在于
PcDNA3质粒中;阳性重组子进行基因测序，证明插入PcDNA3载体中的墓
因片段与genebar水中J型C .t的 omPA基因序列一致。上述结果证明本实验
成功构建了C.tJ型野生株。mpA基因的重组子ompA一peDNA3。
Chlamydia is an obligate , intracellular, Gram negative bacterial pathogen. The diseases evoked by chlamydia are quite wide. In developed countries, the pathogen is the leading cause of sexually transmitted disense. Infection in the eyes results in trachoma which once spreaded widely in the whole world. By now trachoma is still the leading cause of preventable blindness in the world. Chlamydia infection can also make cattle and sheep abort, hens not lay eggs normally. So chlamydia has made huge damage to both mankind and anminals. Chlamydia has became the important pathogen which absorbs people's attention. Chlamydial vaccion has the probability of controling chlamydial infection and spread. People have studied chlamydia vaccine for more then 50 years. Many kinds of vaccine have been tested, such as the inactivated pathogen vaccine, the live attenuated pathogen vaccine, the subunit vaccine, the dendritic cell vaccine and so on. But there isa't a mature vaccine that can be used now. In recent years, the fast
and deep research OB the DNA vaccine brings new hope for stydying the chlamydia DNA vaccine.
The DNA vaccine, which is a recombinant of plasmid with a gene fagment encoding antigen protein, vaccinate the organism directly , transform the organism cells, express the protective antigen, and then induce the organism to obtain specefic immunity.
Our test is aming at constructing an recombinant of ompA gene from the chlamydia trachomatis wild strain. We choose pcDNA3 plasmid as the vector and ompA gene which encodes chlamydia trachomatis (C.t) major outer membrane protein as the target DNA. Clinical chlamydia trachomatis samples,urethral and cervical secreations, were collected from persons who presented to the outpatient department of the sexually transmitted disease. After lysised, clinical samples wert used as template for polymerase chain reaction(PCR). Firstly ,to select C.t positive
samples, we amplified ompA gene through PCR using C.t species specific primier, and then we ascertain the C.t clinical specimen's serotype by the DNA sequence method. Secondly, through polymerase chain reaction, we amplified specific serotype C.t ompA gene using serotype specific primier. Then we enzymatically inserted DNA fragment into plasmid vector, we used three methods to screen positive recombinant, including culturing the E.coli on LB agarose plate containing ampicillin, amplifing ompA gene from recomibant through the PCR method, and digesting recombinant by restriction endonuclease. Finally, we sequenced the inserted DNA fragment by sending the positive recombinant to the sequence corporation.
The results are as follows. By using the PCR method to screen positive C.t clinical samples, the obtained DNA fragment is about 1.2Kb long. The sample which was sended to be sequenced was proved to be C.t J serotype. Agarose gel electrophoresis manifests that the amplified C.t J serotype ompA gene is a little longer than 1.0Kb. The length of the amplified DNA fragment is consistent whih what we have anticipated. When positive recombinant is screened by the PCR method, agarose gel electrophoresis manifests that ompA gene lies in pcDNA3 plasmid. Digested recombinant with restriction endonuclease, we obtained two DNA fragments of separately 5.4Kb and 1.0Kb. The result also confirms that the ompA gene lies in pcDNA3 plasmid. When we sequenced the inserted DNA fragment, the result proved that the inserted gene has the same sequence with C.t J serotype ompA gene provided by genebank. Above results manifest that our test have successfully construsted an recombinant of ompA gene from chlamydia trachomatis wild strai
n J serotype.
引文
[1]Frick KD, Colchero MA, Dean D. Modeling the economic benefit of a potential vaccination program against ocular infection with chlamydia trachomatis. Vaccine, 2004;22(5-6):689
[2]Su Hua, Messer Ronald, Whitmir William, et al.Vaccination against chlamydial genital tract infection after immunization with dendritic cells pulsed ex vivo with nonviable chlamydiae. The Journal of Experimental Medicine, 1998;188(5):809
[3]Shaw Jennifer, Gnmd Vemon, Durling Luke, et al.Dendritic cells pulsed with a recombinant ehlamydial major outer membrane protein antigen elicit a CD4(+)type 2 rather than type 1 immune response that is not protective. Infect Immune,2002;70(3): 1097
[4]Heath C.B.,Heath J.M..Chlamydia trachomatis infection update .Am. Fam. Physician, 1995;52:1455
[5]Holder D.W., Woods E.R..Chlamydia trachomatis screening in the adolescent population. Curr Opin Pediatr, 1997; 9:317
[6]Krause W,Bohdng C.Male infertility and genital chlamydial infection: victim or perpetrator.Andrologia,2003 ;35(4):209;
[7]刘全忠主编《衣原体与衣原体疾病》天津科学科技书出版社2004年
[8]Debattista J,Timms P,Allan J,et al.Immunopathogenesis of Chlamydia trachomatis infections in women.Fertil Steril,2003;79(6): 1273
[9]W. Beagley a,b, Peter Timms c, Chlamydia trachomatis infection: incidence, health costs and prospects for vaccine Development Kenneth .Journal of Reproductive Immunology, 2000;(48):47
[10]Frick KD, Colehero MA, Dean D. Modeling_ the economic net benefit of a potential vaccination program against ocular infection with chlamydia trachomatis. Vaccine,2004;22(5-6):689
[11]Basinkevich AB,Shakhovich RM,Martynova VR, et al.Role of chlamydia, mycoplasma and cytomegalovuirus infention in the development of coronary artery disease. Kardiologiia, 2003; 43(11):4
[12]徐万祥 沙眼衣原体的免疫清除机制及其疫苗制各国外医学计划生育分册2002，(21)1：47
[13]de la Maza, M.A.and de la Maza L.M.A new computer model for estimating the impact of vaccination protocols and its application to the study of Chlamydia traehomatis genital infections, vaccine, 1995;13:119
[14]陈陆源主编《微生物学》人民卫生出版社2002年
[15]Rank R.G., Batteiger B.E.. Protective role of serum antibody in immunity to chlamydial genital infection.Infect Immun,1989;57:299
[16]Moore Terri,Ananaba Godwin A.,Bolier Jacqueline, et al.Fc receptor regulation of protective immunity against chlamydia trachomatis. Immunology, 2002;105(2):213
[17]Su H., Feilzer K., Caldwell H.D.,et al. Chlamydia trachomatis genital tract infection of antibody deficient gene knockout mice. Infect Immun. 1997;65: 1993
[18]Williams, D.M. Humoral and cellular immunity in secondary infection due to murine chlamydia trachomatis. Infect Immun, 65:2876
[19]Richard S Stephens.Chlamydial genomics and vaccine antigen discovery.The Journal of In.fectious Diseases, 2000; 181 :S521
[20]Berry LJ, Hickey DK, Skelding KA,et al.Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against chlamydia muridarum genital tract infection. Infect Immun,2004; 72(2): 1019
[21]S.U,Messer.R, Whitmire.W.Subclinical chlamydia infection of the female mouse genital tract generates a potent protective immune response : implications for development of live attenuated ehlamydial vaccine strains.Infect Immun, 2000; 192
[22]Sukumar Pal,Ida Theodor, Ellena M Peterson, et al.Immunization with an acellular vaccine consisting of the outer membrane complex of chlamydia trachomatis induces protection against a genital challenge Infect Immun, 1997;3361
[23]Lichtenwalner A.B, Patton DL, Van Voorhis WC,et al.Heat shock protein 60 is the major antigen which stimulates delayed type hypersensitivity reaction in the macaque model of chlamydia trachomatis salpingitis. Infect Immun, 2004;72(2): 1159
[24]Andrie R, Braun P, Welsch U,et al.Chlamydial and human heat shock protein 60 homologues in acute coronary syndromes. (Auto)immune reactions as a link between infection and atherosclerosis. Z Kardiol,2003;92(6):455
[25]Xu Q.Infections, heat shock proteins, and atherosclerosis. Curr Opin Cardiol, 2003;18(4):245
[26]Batteiger B.E.,Rank R.G.,et al.Partial protection against genital reinfection by immunization of guinea pigs with isolated outer membrane proteins of the ehlamydial agent of guinea pig inclusion conjunctivitis. J. Gen Microbiol, 1993; 139:2965
[27]Tuffrey M. Heterotypic protection of mice against chlamydial salpingitis and colonization of the lower genital tract with the human servar 'F isolate of chlamydia trachomatis by prior immunization with recombinant serovar L1 major outer membrane protein.J Gen Microbiol, 1992; 138:1707
[28]Su H., Parnell M.,Caldwell H.D. Protective efficacy of a parenter ally
administered MOMP derived synthetic oligopeptide vaccine in a mudne model of Chlamydia trachomatis genital tract infection: serum neutralizing IgG antibodies do not protect against chlamydial genital tract infection.Vaccine, 1995; 13:1023
[29]Campos M. A ehlamydial major outer membrane protein extract as a trachoma vaccine candidate. Invest Ophthalmol Vis Sci. 1995: 36:1477
[30]陈启民 王金忠 耿运琪 主编《分子生物学》南开大学出版社2001年
[31]Frost EH.Typing ehlamydia trachomatis by election of restriction fragment length polymorphism in the gene encoding the major outer membrane protein.J Infect Disease, 1991; 163:1103
[32]卢圣栋主编《现代分子生物学实验技术》 高等教育出版社
[33]WOL FF J A, MALONE R W, WILL IAMS P ,et al.Direet gene transfer into mouse muscle in vivo.J Science, 1990;247:1465
[34]Kumar H, Malhotra D, Goswami S.How far have we reached in tuberculosis vaccine development? Crit Rev Microbiol, 2003; 29(4):297
[35]Sugawara I, Yamada H, Udagawa T. Vaccination of guinea pigs with DNA encoding Ag85A by gene gun bombardment. Tuberculosis (Edinb),2003;83(6):331
[36]Back KM, Ko SY, Lee M,et al. Comparative analysis of effects of cytokine gene adjuvants on DNA vaccination against Mycobacterium tuberculosis heat shock protein 65. Vaccine,2003 ;21 (25-26):3684
[37]Miyaji EN, Dias WO, Tanizaki MM, et al.Proteetive efficacy of PspA (pneumoeoeeal surface protein A) based DNA vaccines: contribution of both humoral and cellular immune responses. FEMS Immunol Med Microbiol, 2003;37(1):53
[38]Lemieux P.Teehnological advances to increase irnmunogenicity of DNA vaccines. Expert Rev Vaccines.2002:1 (1):85
[39]周宗安等．基因疫苗的研究进展及临床应用．东南国防医药，2004；(5)2：99
[40]Andrew D,Murdin,Hua Su,Michel H.Polivirus hybirds expression nutralization epitopes from varible domains Ⅰ and Ⅳ of the major outer membrane protein of chlamydia trachomatis elicit broadly cross Reactive etraehomatis nutrilizing antibodies. Infect Immun, 1995; 1161
[41]Dong Ji Zhang,Xi Yang,Jody Berry, et al.DNA vaccination with the major outer membrane protein gene induces acquired immunity to chlamydia trachomatis(Mouse Pneumonitis)Infection.The Journal of Infectious Diseases :.1997;176:1035
[42]Brtmham RC , Zhang DJ . Transgene as vaccine for chlamydia. American Heart Journal, 1999; s519
[43]Héchard Céline,Grépinet Olivier, Rodolakis Annie.Evaluation of protection against chlamydophila abortus challenge after DNA immunization with the major outer membrane protein encoding gene in pregnant and non-pregnant mice.The Journal of Medical Microbiology,2003 ;52(1):35
[44]Donati M, Sambri V, Comanducci M,et al.DNA immunization with pgp3 gene of Chlamydia trachomatis inhibits the spread of chlarnydial infection from the lower to the upper genital tract in C3H/HeN mice. Vaccine,2003 ;21 (11-12): 1089
[45]Johansson M, Sehon K, Ward M ,et al. Genital tract in.fection with chlamydia trachomatis fails to induce protective immunity in gamma interferon receptor deficient mice despite a strong local immunoglobulin A response. Infect Immun, 1997; 65:1032
[46]Murdin AD, Durra P, Sodoyer R ,et al. Use of a mouse lung challenge model to identify antigens protective against chlamydia pneumoniae lung infection. J Infect Dis, 2000; 181 (Suppl. 3):S544
[47]Svanholm C, Bandholtz L, Castanos Velez E ,et al. Protective DNA immunization against chlamydia pneumoniae. Stand J Immunol, 2000; 51:345
[48]Sukumar Pal,Kerry M.Bamhart,Qun Wei,et al.Vaccination of mice with DNA plasmids coding for the chlamydia trachomatis major outer membrane protein elicits an immune response but fails to protect against a genital challenge.Vaccine, 1999(17):459
[49]Newhall WJ,Batteiger VB,Johns RB.Analysis of the human serological resoponse to proteins of chlamydia trachomatis.Infect Immune, 1982;32:1181
[50]Chun Lin Yang,lan Maclean,Robert C Brunham.DNA sequence polymorphism of the chlamydia trachomatis ompA gene.The Journal of Infectious Disease, 1993;168:1225
[51]Diane E Kawa,Richard S Stephens.Antigentic topology of chlamydial PorB protein and identification of targets for immune nutralization of Infectivity.The Journal of Immunology, 2002; 168:5184
[52]Kubo A, R S Stephens.Characterization and functional analysis of PorB,a Chlarnydia porin and neutralizing target. Mol Microbiol, 2000;(38):722
[53]Fling SP, Sutherland RA,Steele LN,et aI.CD8~+ T cells recognize an inclusion membrane associated protein from the vacuolar pathogen chlamydia traehomatis.Proe Natl Acad Sci U S A, 2001;98(3): 1160
[54]Burnham RC,Zhang DJ,Yang X. The potential for development against chlamydial infection and disease. The Journal of infectious, 2000; 181 (supp13):s538
[55]Lu H,Xing Z,Brunham RC.GM-CSF transgene based adjuvant allows the
establishment of protective mucosal immunity following vaccination with inactivated chlarnydia trachomatis. Immunol, 2002; 1169
[56]Pal S,Davis HL,Peterson EM, et al.Immunization with the chlamydia traehomatis mouse pneumonitis major outer membrane protein by use of CpG oligodeoxynucleotides as an adjuvant imduces a protective immune response against an intranasal chlamydial challenge.Infect Immune, 2002;70(9):4812
[57]Sukumar Pal,Catherine J Luke,Alan G Barbour, et al.Immunization with the chlamydia trachomatis major outer membrane protein, using the outer surface protein A of Borrelia burgdorferi as an adjuvant, can induce protection against a chlamydial genital challenge. Vaccine,2003 ;21 (13-14): 1455
[58]L Bandholtz, M R Kreuger, C Svanholm, et al.Adjuvant modulation of the immune responses and the outcome of infection with chlamydia pneumoniae. Clin Exp Immunol ,2002; 130:393
[59]Yukio Sato,Mark Roman,Helen Tighe ,et al.Immunostimulatory DNA necessary for effective intradermal gene immunization. Science, 1996; 275:352
[60]Riemenschneider J, Garrison A, Geisbert J.Comparison of individual and combination DNA vaccines for B. anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus. Vaccine, 2003;21(25-26):4071
[2]Su Hua, Messer Ronald, Whitmir William, et al.Vaccination against chlamydial genital tract infection after immunization with dendritic cells pulsed ex vivo with nonviable chlamydiae. The Journal of Experimental Medicine, 1998;188(5):809
[3]Shaw Jennifer, Gnmd Vemon, Durling Luke, et al.Dendritic cells pulsed with a recombinant ehlamydial major outer membrane protein antigen elicit a CD4(+)type 2 rather than type 1 immune response that is not protective. Infect Immune,2002;70(3): 1097
[4]Heath C.B.,Heath J.M..Chlamydia trachomatis infection update .Am. Fam. Physician, 1995;52:1455
[5]Holder D.W., Woods E.R..Chlamydia trachomatis screening in the adolescent population. Curr Opin Pediatr, 1997; 9:317
[6]Krause W,Bohdng C.Male infertility and genital chlamydial infection: victim or perpetrator.Andrologia,2003 ;35(4):209;
[7]刘全忠主编《衣原体与衣原体疾病》天津科学科技书出版社2004年
[8]Debattista J,Timms P,Allan J,et al.Immunopathogenesis of Chlamydia trachomatis infections in women.Fertil Steril,2003;79(6): 1273
[9]W. Beagley a,b, Peter Timms c, Chlamydia trachomatis infection: incidence, health costs and prospects for vaccine Development Kenneth .Journal of Reproductive Immunology, 2000;(48):47
[10]Frick KD, Colehero MA, Dean D. Modeling_ the economic net benefit of a potential vaccination program against ocular infection with chlamydia trachomatis. Vaccine,2004;22(5-6):689
[11]Basinkevich AB,Shakhovich RM,Martynova VR, et al.Role of chlamydia, mycoplasma and cytomegalovuirus infention in the development of coronary artery disease. Kardiologiia, 2003; 43(11):4
[12]徐万祥 沙眼衣原体的免疫清除机制及其疫苗制各国外医学计划生育分册2002，(21)1：47
[13]de la Maza, M.A.and de la Maza L.M.A new computer model for estimating the impact of vaccination protocols and its application to the study of Chlamydia traehomatis genital infections, vaccine, 1995;13:119
[14]陈陆源主编《微生物学》人民卫生出版社2002年
[15]Rank R.G., Batteiger B.E.. Protective role of serum antibody in immunity to chlamydial genital infection.Infect Immun,1989;57:299
[16]Moore Terri,Ananaba Godwin A.,Bolier Jacqueline, et al.Fc receptor regulation of protective immunity against chlamydia trachomatis. Immunology, 2002;105(2):213
[17]Su H., Feilzer K., Caldwell H.D.,et al. Chlamydia trachomatis genital tract infection of antibody deficient gene knockout mice. Infect Immun. 1997;65: 1993
[18]Williams, D.M. Humoral and cellular immunity in secondary infection due to murine chlamydia trachomatis. Infect Immun, 65:2876
[19]Richard S Stephens.Chlamydial genomics and vaccine antigen discovery.The Journal of In.fectious Diseases, 2000; 181 :S521
[20]Berry LJ, Hickey DK, Skelding KA,et al.Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against chlamydia muridarum genital tract infection. Infect Immun,2004; 72(2): 1019
[21]S.U,Messer.R, Whitmire.W.Subclinical chlamydia infection of the female mouse genital tract generates a potent protective immune response : implications for development of live attenuated ehlamydial vaccine strains.Infect Immun, 2000; 192
[22]Sukumar Pal,Ida Theodor, Ellena M Peterson, et al.Immunization with an acellular vaccine consisting of the outer membrane complex of chlamydia trachomatis induces protection against a genital challenge Infect Immun, 1997;3361
[23]Lichtenwalner A.B, Patton DL, Van Voorhis WC,et al.Heat shock protein 60 is the major antigen which stimulates delayed type hypersensitivity reaction in the macaque model of chlamydia trachomatis salpingitis. Infect Immun, 2004;72(2): 1159
[24]Andrie R, Braun P, Welsch U,et al.Chlamydial and human heat shock protein 60 homologues in acute coronary syndromes. (Auto)immune reactions as a link between infection and atherosclerosis. Z Kardiol,2003;92(6):455
[25]Xu Q.Infections, heat shock proteins, and atherosclerosis. Curr Opin Cardiol, 2003;18(4):245
[26]Batteiger B.E.,Rank R.G.,et al.Partial protection against genital reinfection by immunization of guinea pigs with isolated outer membrane proteins of the ehlamydial agent of guinea pig inclusion conjunctivitis. J. Gen Microbiol, 1993; 139:2965
[27]Tuffrey M. Heterotypic protection of mice against chlamydial salpingitis and colonization of the lower genital tract with the human servar 'F isolate of chlamydia trachomatis by prior immunization with recombinant serovar L1 major outer membrane protein.J Gen Microbiol, 1992; 138:1707
[28]Su H., Parnell M.,Caldwell H.D. Protective efficacy of a parenter ally
administered MOMP derived synthetic oligopeptide vaccine in a mudne model of Chlamydia trachomatis genital tract infection: serum neutralizing IgG antibodies do not protect against chlamydial genital tract infection.Vaccine, 1995; 13:1023
[29]Campos M. A ehlamydial major outer membrane protein extract as a trachoma vaccine candidate. Invest Ophthalmol Vis Sci. 1995: 36:1477
[30]陈启民 王金忠 耿运琪 主编《分子生物学》南开大学出版社2001年
[31]Frost EH.Typing ehlamydia trachomatis by election of restriction fragment length polymorphism in the gene encoding the major outer membrane protein.J Infect Disease, 1991; 163:1103
[32]卢圣栋主编《现代分子生物学实验技术》 高等教育出版社
[33]WOL FF J A, MALONE R W, WILL IAMS P ,et al.Direet gene transfer into mouse muscle in vivo.J Science, 1990;247:1465
[34]Kumar H, Malhotra D, Goswami S.How far have we reached in tuberculosis vaccine development? Crit Rev Microbiol, 2003; 29(4):297
[35]Sugawara I, Yamada H, Udagawa T. Vaccination of guinea pigs with DNA encoding Ag85A by gene gun bombardment. Tuberculosis (Edinb),2003;83(6):331
[36]Back KM, Ko SY, Lee M,et al. Comparative analysis of effects of cytokine gene adjuvants on DNA vaccination against Mycobacterium tuberculosis heat shock protein 65. Vaccine,2003 ;21 (25-26):3684
[37]Miyaji EN, Dias WO, Tanizaki MM, et al.Proteetive efficacy of PspA (pneumoeoeeal surface protein A) based DNA vaccines: contribution of both humoral and cellular immune responses. FEMS Immunol Med Microbiol, 2003;37(1):53
[38]Lemieux P.Teehnological advances to increase irnmunogenicity of DNA vaccines. Expert Rev Vaccines.2002:1 (1):85
[39]周宗安等．基因疫苗的研究进展及临床应用．东南国防医药，2004；(5)2：99
[40]Andrew D,Murdin,Hua Su,Michel H.Polivirus hybirds expression nutralization epitopes from varible domains Ⅰ and Ⅳ of the major outer membrane protein of chlamydia trachomatis elicit broadly cross Reactive etraehomatis nutrilizing antibodies. Infect Immun, 1995; 1161
[41]Dong Ji Zhang,Xi Yang,Jody Berry, et al.DNA vaccination with the major outer membrane protein gene induces acquired immunity to chlamydia trachomatis(Mouse Pneumonitis)Infection.The Journal of Infectious Diseases :.1997;176:1035
[42]Brtmham RC , Zhang DJ . Transgene as vaccine for chlamydia. American Heart Journal, 1999; s519
[43]Héchard Céline,Grépinet Olivier, Rodolakis Annie.Evaluation of protection against chlamydophila abortus challenge after DNA immunization with the major outer membrane protein encoding gene in pregnant and non-pregnant mice.The Journal of Medical Microbiology,2003 ;52(1):35
[44]Donati M, Sambri V, Comanducci M,et al.DNA immunization with pgp3 gene of Chlamydia trachomatis inhibits the spread of chlarnydial infection from the lower to the upper genital tract in C3H/HeN mice. Vaccine,2003 ;21 (11-12): 1089
[45]Johansson M, Sehon K, Ward M ,et al. Genital tract in.fection with chlamydia trachomatis fails to induce protective immunity in gamma interferon receptor deficient mice despite a strong local immunoglobulin A response. Infect Immun, 1997; 65:1032
[46]Murdin AD, Durra P, Sodoyer R ,et al. Use of a mouse lung challenge model to identify antigens protective against chlamydia pneumoniae lung infection. J Infect Dis, 2000; 181 (Suppl. 3):S544
[47]Svanholm C, Bandholtz L, Castanos Velez E ,et al. Protective DNA immunization against chlamydia pneumoniae. Stand J Immunol, 2000; 51:345
[48]Sukumar Pal,Kerry M.Bamhart,Qun Wei,et al.Vaccination of mice with DNA plasmids coding for the chlamydia trachomatis major outer membrane protein elicits an immune response but fails to protect against a genital challenge.Vaccine, 1999(17):459
[49]Newhall WJ,Batteiger VB,Johns RB.Analysis of the human serological resoponse to proteins of chlamydia trachomatis.Infect Immune, 1982;32:1181
[50]Chun Lin Yang,lan Maclean,Robert C Brunham.DNA sequence polymorphism of the chlamydia trachomatis ompA gene.The Journal of Infectious Disease, 1993;168:1225
[51]Diane E Kawa,Richard S Stephens.Antigentic topology of chlamydial PorB protein and identification of targets for immune nutralization of Infectivity.The Journal of Immunology, 2002; 168:5184
[52]Kubo A, R S Stephens.Characterization and functional analysis of PorB,a Chlarnydia porin and neutralizing target. Mol Microbiol, 2000;(38):722
[53]Fling SP, Sutherland RA,Steele LN,et aI.CD8~+ T cells recognize an inclusion membrane associated protein from the vacuolar pathogen chlamydia traehomatis.Proe Natl Acad Sci U S A, 2001;98(3): 1160
[54]Burnham RC,Zhang DJ,Yang X. The potential for development against chlamydial infection and disease. The Journal of infectious, 2000; 181 (supp13):s538
[55]Lu H,Xing Z,Brunham RC.GM-CSF transgene based adjuvant allows the
establishment of protective mucosal immunity following vaccination with inactivated chlarnydia trachomatis. Immunol, 2002; 1169
[56]Pal S,Davis HL,Peterson EM, et al.Immunization with the chlamydia traehomatis mouse pneumonitis major outer membrane protein by use of CpG oligodeoxynucleotides as an adjuvant imduces a protective immune response against an intranasal chlamydial challenge.Infect Immune, 2002;70(9):4812
[57]Sukumar Pal,Catherine J Luke,Alan G Barbour, et al.Immunization with the chlamydia trachomatis major outer membrane protein, using the outer surface protein A of Borrelia burgdorferi as an adjuvant, can induce protection against a chlamydial genital challenge. Vaccine,2003 ;21 (13-14): 1455
[58]L Bandholtz, M R Kreuger, C Svanholm, et al.Adjuvant modulation of the immune responses and the outcome of infection with chlamydia pneumoniae. Clin Exp Immunol ,2002; 130:393
[59]Yukio Sato,Mark Roman,Helen Tighe ,et al.Immunostimulatory DNA necessary for effective intradermal gene immunization. Science, 1996; 275:352
[60]Riemenschneider J, Garrison A, Geisbert J.Comparison of individual and combination DNA vaccines for B. anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus. Vaccine, 2003;21(25-26):4071