阴道毛滴虫病毒基因组全长cDNA克隆
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摘要
本研究首先从临床分离阴道毛滴虫,用肝浸汤进行纯培养,提取虫体总核酸,1%琼脂糖凝胶电泳,初步筛选出一株携带阴道毛滴虫病毒的虫株;然后根据国外已发表的阴道毛滴虫序列设计了一对覆盖全长的特异性引物,对阴道毛滴虫病毒基因组进行了全长cDNA克隆并测序,测得我国阴道毛滴虫病毒基因组全长为4649bp。该序列编码两个阅读框(ORF),二个阅读框有14nt的重复,在重复区有一个7聚体的滑动体(C CTT TTT)。ORF1(nt290-2326)包含678个氨基酸,编码分子量为74.9KDa的病毒衣壳蛋白;ORF2(nt2310-4581)包含756个氨基酸,编码分子量为85.8KDa的RNA依赖的RNA聚合酶(RDRP)蛋白。本研究的结果为深入研究我国阴道毛滴虫病毒奠定了良好基础。
Trichomonas vaginalis, which parasitizes in urogenital system, is one kind of flagellates causing vaginal inflammation. At present, the characterization of T.vaginalis has not been understood completely and the discovery of Trichomonas vaginalis virus(TVV) played a pivotal role for T.vaginalis study in molecular biology. Several papers suggested that there were different kinds of dsRNA viruses in T.vaginalis. The T.vaginalis with virus was isolated from patients in Changchun and culture was carried out in vitro, the full-length cDNA of Trichomonas vaginalis virus was sequenced and constructed.
T.vaginalis cultured in vitro T.vaginalis isolated from Changchun patients were cultured successfully in liver medium in vitro. The T.vaginalis strain with virus which had a 5.5kb extra band except genomic DNA was selected by 1% agarose gel electrophoresis analysis on total nucleic acids extracted from T. vaginalis. The T.vaginalis strain selected was cultured under axenic condition. The body configuration of paradises was observed by light and electron microscope. Growth state was very well in ox liver medium (pH=5.54) and the vital force didn’t decrease after 50 generation. The strain freezed in 10% DMSO was cultured continuously after recovery and the
Trichomonas vaginalis, which parasitizes in urogenital system, is one kind of flagellates causing vaginal inflammation. At present, the characterization of T.vaginalis has not been understood completely and the discovery of Trichomonas vaginalis virus(TVV) played a pivotal role for T.vaginalis study in molecular biology. Several papers suggested that there were different kinds of dsRNA viruses in T.vaginalis. The T.vaginalis with virus was isolated from patients in Changchun and culture was carried out in vitro, the full-length cDNA of Trichomonas vaginalis virus was sequenced and constructed.
T.vaginalis cultured in vitro T.vaginalis isolated from Changchun patients were cultured successfully in liver medium in vitro. The T.vaginalis strain with virus which had a 5.5kb extra band except genomic DNA was selected by 1% agarose gel electrophoresis analysis on total nucleic acids extracted from T. vaginalis. The T.vaginalis strain selected was cultured under axenic condition. The body configuration of paradises was observed by light and electron microscope. Growth state was very well in ox liver medium (pH=5.54) and the vital force didn’t decrease after 50 generation. The strain freezed in 10% DMSO was cultured continuously after recovery and the
引文
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[2] Street D A, Robinson D T and Hetherington C M. Infection of female squirrel monkey (saimiri sciureus) with Trichomonas vaginalis as a model of trichomoniasis in women[J]. Br.J.Vener.Dis, 1983, 59: 249~254.
[3] McGrory T and Garber G E. Mouse intravaginal infection with Trichomonas vaginalis and role of Lactobacillus acidophilus in sustaining infection[J]. Infection Immun, 1992, 60: 2375~2379.
[4] Rein and Muller. Trichomonas vaginalis in Sexually transmitted disease[J]. Holmesed Mcgraw-Hill, 1984, 525~536.
[5] Tuttle J P, Holbrook T W and Derrick F C. Interference of the human spermatozoal motility by Trichomonas vaginalis[J].Urol, 1977, 118:102~109.
[6] Cotch M F, Pastorek J G, Nugent R P, et al. Demographic and behavioral mediators of Trichomonas vaginalis infection among pregnant women[J].Obstet Gynecol, 1991, 78: 1087~1092.
[7] Niccolai L M, Kopicko J J, Kassie A, et al. Incidence and predictors of reinfection with Trichomonas vaginalis in HIV-infected women [J].SexTransmDis, 2000, 27(5): 284~288.
[8] 马济宏, 周全, 高进兰, 等. 阴道毛滴虫在性传播疾病患者寄生情况的研究[J].中国寄生虫病防治杂志, 1999, 12(2): 108~109 .[9] 陈佩惠. 人体寄生虫学(第四版)[M]. 北京:人民卫生出版社, 1997, 52~53.
[10] 叶 英, 黄美玉, 连惟能. 阴道毛滴虫的研究 X 阴道毛滴虫电子显微镜观察的初步报告[J].上海第一医学院学报, 1964, 2(2): 228 ~230
[11] 杜之鸣, 周文琴, 韩文素, 等. 阴道毛滴虫超微结构的进一步观察[J].中国寄生虫学与寄生虫病杂志, 1990, 8: 47.
[12] Nielsen M H and Nielsen R. Electronmicroscopy of Trchomonas vaginalis donneinter action with vaginal epitheliumin human trichomoniasis[J]. Acta Path Microbiol Scand Sect B, 1975, 83: 305.
[13] 陈文列, 钟秀容, 陈连云, 等. 阴道毛滴虫的超微结构细胞化学研究[J]. 电子显微学报, 1994, 5: 346.
[14] DemirezenS ,S afiZ .The interaction of trichomonas with epithelial cell, polymorphonuclear leucocytes and erythrocytes on vaginal smears:light microscopic obser vation[J]. Cytopathology,2000,11(5):326一332. [15」刘永春,郭永和,王冬梅.男性不育精液阴道毛滴虫检测结果分析[J]. 男科学报,1998,4(1):51-52. [16」山长武,李静.阴道毛滴虫与慢性前列腺炎关系的研究[J] .中国寄生虫病防治杂志,1997,10(4):317.
[17] Gilbert R O, Elia G, Beacch D H, et al. Cytopathogentic effect of Trichomonas vaginalis on human vaginal epithelial cells cultured in vitro[J]. Infect Immun, 2000, 68(7): 4200~4206.
[18] van Der Schee C, van Belku M A, Zwijgers L, et al. Improved diagnosis of Trichomonas vaginalis infection by PCR using vaginal swabs and urine specimens compared to diagnosis by wetmount microscopy, culture, and fluorescent staining[J]. Clin Microbiol, 1999, 37(12): 4127~4130.
[19] Riley D E, Roberts M C, Takayama T, et al. Development of apolymerase chain reaction based diagnosis of Trichomonas vaginalis. [J].Clin Microbiol, 1992, 30(2): 465~472.
[20] Schwebke J R, Venglarik M F and Morgan S C. Delayed versus immediate beside inoculation of culture media for diagnosis of vaginal trichomonas[J]. Clin Microbiol,1999, 37(7): 2369~2370.
[21] 唐玉钗,冯修高,兰小鹏,等.一份敏感的浚化胶乳阴道毛滴虫凝集实验(附486份报告)[J].福建医药杂志,1998 ,2 0( 3 ): 10 9一110.
[22] 祝虹,高兴政,朱永红.用酶联免疫吸附实验双抗体夹心法检测阴道毛滴虫可溶性抗原的研究〔J].中国寄生虫病防治杂志 , 1998,11(3):177-1 79.
[23] 张跃,申厚凤,杨维平,等.应用斑点免疫实验检测阴道毛滴虫抗原 [J].江苏临床医学杂志,1997,1(2):82.
[24] MaytaH ,G ilmanR H,C alderonM M,et al. 18S ribosom al D N A –based PCR ford iagnosis of Trichontonas vaginalis [J]. J Clin Microbiol,2000,38(7):2683一2687.
[25] Gelbart S M, Thomason J L, Osypowski P J, et al. Growth of Trichomonas vaginalis in commercial culture media[J]. Clin Microbiol, 1990, 28: 962~964.
[26] Levi M H, Torres J, Pina C, et al. Comparison of the In Pouch TV culture system and Diagond’s modified medium for detection of Trichomonas vaginalis[J]. Clin Microbiol, 1997, 35: 3308~3310.
[27] Stary A, Kuchinka Koch A and Teodorowicz L. Detection of Trichomonas vaginalis on modified Columbiaagar in the routine laboratory[J]. Clin Microbiol, 2002, 40: 3277~3280.
[28] Madico G, Quinn T C, Rompalo A, et al. Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples[J]. Clin Microbiol, 1998, 36(11): 3205~3210.
[29] Alderete J F, and Garza, W E. Identication and properties of Trichomonas vaginalis proteins involved in cytoadherence[J].Infect Immun, 1988, 56: 28~33.
[30] Wendel K A, Erbelding E J, Gaydos C A, et al. Trichomonas vaginalis polymerase chain reaction compared with standard diagnosistic and the rapeutic protocols for detection and treatment of vaginal trichomoniasis[J]. Clin Infect Dis, 2002, 35:576~580.
[31] 郝敏,高瑜 . 几种常见阴道 炎 的诊治 进 展 [J]. 中 国实用妇科与产科杂志,2005,21(3);134~136.
[32] Sobel JD,Nyirjesy P,BrownW. Tinidazole therapy formetronidazole resistant vaginal trichomoniasis[J]. Clin InfectDis, 2001, 33 (8) : 1341-1346.
[33] Kigozi GG, Brahmbhatt H,Wabwire Mangen F, et al. Treatment of Trichomonas in pregnancy and adverse outcomes of pregnancy: asubanalysis of a randomized trial in Rakai, Uganda. Am J ObstetGynecol, 2003, 189 (5) : 1398-1400.
[34] 苏庆平,沙人珏,李舜华,等.阴道毛滴虫形态结构、染色体及生殖的观察[J].福建医学院学报,1988,22:121.
[35] 王聪敏,胡文英,周 全.吡喹酮对体外培养阴道毛滴虫作用研究[J].南通医学院学报,1987,7(1):12.
[36] 何玉良,陶贤梅,关立华,等.甲硝唑酯体外抗阴道毛滴虫和阿米巴的效果[J].中国寄生虫学与寄生虫病杂志,1995,13:32.
[37] 黄美玉,连惟能.替硝唑体外抗毛滴虫效果观察[J].中国寄生虫学与寄生虫病杂志,1987,5:29.
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