阴道毛滴虫病毒转染载体的构建及EGFP在阴道毛滴虫内的表达
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摘要
本研究根据我国阴道毛滴虫病毒基因组的序列特征,借鉴以往原虫病毒载体构建的研究经验,用绿色荧光蛋白编码基因替换TVV全部或部分编码区,构建阴道毛滴虫病毒转染载体pTVVL289/EGFP、pTVVL289/EGFP/TVVS、pTVVL559/EGFP/TVVS。
阴道毛滴虫病毒转染载体经T7RNA聚合酶体外转录成mRNA后,经电穿孔法转染含TVV的阴道毛滴虫细胞内,用荧光显微镜观察转染后不同时间内EGFP的表达情况。结果表明:载体pTVVL289/EGFP的体外转录体转染阴道毛滴虫后未观察到荧光信号,表明在TVV的3’末端UTR存在有与病毒复制和/或包装有关的序列。载体pTVVL289/EGFP/TVVS、pTVVL559/EGFP/TVVS体外转录体转染后可检测到绿色荧光信号,并在持续传代培养5代后仍不减弱;提取转染后虫体的总核酸,用RT-PCR检测到EGFP的mRNA的存在;SDS-PAGE分析转染虫体的培养上清和虫体中EGFP的表达,检测到大小为27KD的蛋白。说明我们构建的阴道毛滴虫病毒载体可以应用于介导外源基因在阴道毛滴虫体内的表达。
本研究构建了阴道毛滴虫病毒转染载体,介导了目的基因EGFP在阴道毛滴虫体内的表达,为深入研究我国阴道毛滴虫病毒与虫体及其宿主之间的关系提供了新的研究工具,为进一步研究阴道毛滴虫的分子生物学特性奠定基础。
Trichomonas vaginalis, which parasitizes in urogenital system, is one kind of flagellates causing vaginal inflammation. The patients run a six-fold higher risk of HIV infection. Trichomonas vaginalis virus(TVV),is a kind of ds RNA virus parasitized in Trichomonas vaginalis,the prior research shows that the existence of TVV has relationship with the invasiveness and Metronidazole resistance of Trichomonas vaginalis. the discovery of Trichomonas vaginalis virus(TVV) shows a new way for T.vaginalis study in molecular biology. At present, the vector system reconstructed from GLV has been used in several area ,for example : expression of exogenous gene, structure and function analysis of G.. lamblia gene. It has been a significant tool for the study of G.. lamblia in molecular biology.While the study about TVV vector is relatively lagged. Chimera of TVV cDNA and enhanced green fluorescent protein (EGFP) were constructed and their in vitro transcript were electroporated into TVV-infected T. vaginalis trophozoites, the control signals of the TVV genome required for expression of foreign gene were preliminary determined according to the expression level of EGFP in the transfected parasites. This transfection system should provide a valuable tool for genetic study of T. vaginalis.
Construction of GCV Transfer Vector According to feature of T. vaginalis virus (TVV) genome in China,a system was developed here for expression of a foreign gene in this organism by flanking the enhanced green fluorescent protein(EGFP) gene with the fragments of TVV positive-strand RNA . Chimera pTVVL289/EGFP、pTVVL289/EGFP/TVVS、pTVVL559/EGFP/TVVS were constructed.
Expression of EGFP mediated by TVV Transcript of the Chimera pTVVL289/EGFP、pTVVL289/EGFP/TVVS and pTVVL559/EGFP/TVVS were synthesized in vitro with T7 RNA polymerase and used to transfect T. vaginalistrophozoites already infected with TVV. The results indicated that chimera pTVVL289/EGFP/TVVS and pTVVL559/EGFP/TVVS appeared fluorescent signal under fluorescence microscope at 488nm, and without attenuating after the fifth passage .We detected the EGFP mRNA by RT-PCR in transfected T. vaginalis and the results of SDS-PAGE demonstrated that EGFP was expressed in T. vaginalis and mainly secreted to medium supernatant. while chimera pTVVL289/EGFP did not appear fluorescent signal under fluorescence microscope, it shows that the 3’UTRs of TVV play a part in replication,translation and/or package of virus particle.
阴道毛滴虫病毒转染载体经T7RNA聚合酶体外转录成mRNA后,经电穿孔法转染含TVV的阴道毛滴虫细胞内,用荧光显微镜观察转染后不同时间内EGFP的表达情况。结果表明:载体pTVVL289/EGFP的体外转录体转染阴道毛滴虫后未观察到荧光信号,表明在TVV的3’末端UTR存在有与病毒复制和/或包装有关的序列。载体pTVVL289/EGFP/TVVS、pTVVL559/EGFP/TVVS体外转录体转染后可检测到绿色荧光信号,并在持续传代培养5代后仍不减弱;提取转染后虫体的总核酸,用RT-PCR检测到EGFP的mRNA的存在;SDS-PAGE分析转染虫体的培养上清和虫体中EGFP的表达,检测到大小为27KD的蛋白。说明我们构建的阴道毛滴虫病毒载体可以应用于介导外源基因在阴道毛滴虫体内的表达。
本研究构建了阴道毛滴虫病毒转染载体,介导了目的基因EGFP在阴道毛滴虫体内的表达,为深入研究我国阴道毛滴虫病毒与虫体及其宿主之间的关系提供了新的研究工具,为进一步研究阴道毛滴虫的分子生物学特性奠定基础。
Trichomonas vaginalis, which parasitizes in urogenital system, is one kind of flagellates causing vaginal inflammation. The patients run a six-fold higher risk of HIV infection. Trichomonas vaginalis virus(TVV),is a kind of ds RNA virus parasitized in Trichomonas vaginalis,the prior research shows that the existence of TVV has relationship with the invasiveness and Metronidazole resistance of Trichomonas vaginalis. the discovery of Trichomonas vaginalis virus(TVV) shows a new way for T.vaginalis study in molecular biology. At present, the vector system reconstructed from GLV has been used in several area ,for example : expression of exogenous gene, structure and function analysis of G.. lamblia gene. It has been a significant tool for the study of G.. lamblia in molecular biology.While the study about TVV vector is relatively lagged. Chimera of TVV cDNA and enhanced green fluorescent protein (EGFP) were constructed and their in vitro transcript were electroporated into TVV-infected T. vaginalis trophozoites, the control signals of the TVV genome required for expression of foreign gene were preliminary determined according to the expression level of EGFP in the transfected parasites. This transfection system should provide a valuable tool for genetic study of T. vaginalis.
Construction of GCV Transfer Vector According to feature of T. vaginalis virus (TVV) genome in China,a system was developed here for expression of a foreign gene in this organism by flanking the enhanced green fluorescent protein(EGFP) gene with the fragments of TVV positive-strand RNA . Chimera pTVVL289/EGFP、pTVVL289/EGFP/TVVS、pTVVL559/EGFP/TVVS were constructed.
Expression of EGFP mediated by TVV Transcript of the Chimera pTVVL289/EGFP、pTVVL289/EGFP/TVVS and pTVVL559/EGFP/TVVS were synthesized in vitro with T7 RNA polymerase and used to transfect T. vaginalistrophozoites already infected with TVV. The results indicated that chimera pTVVL289/EGFP/TVVS and pTVVL559/EGFP/TVVS appeared fluorescent signal under fluorescence microscope at 488nm, and without attenuating after the fifth passage .We detected the EGFP mRNA by RT-PCR in transfected T. vaginalis and the results of SDS-PAGE demonstrated that EGFP was expressed in T. vaginalis and mainly secreted to medium supernatant. while chimera pTVVL289/EGFP did not appear fluorescent signal under fluorescence microscope, it shows that the 3’UTRs of TVV play a part in replication,translation and/or package of virus particle.
引文
[1] Rein and Muller. Trichomonas vaginalis in Sexually transmitted disease[J]. Holmesed Mcgraw-Hill, 1984, 525~536
[2] 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
[3] 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
[4] 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
[5] Mattern CFT,Diamond LS,Daniel TL.Viruses of Entamoeba histolytica. Ⅱ. Morphogenesis of the polyhedral particle (ABRAM2-HK-9)-HB-301 and the filamentous agent (ABRM)2 -HK- 9[J]. J Virol,1972,9:342~358
[6] Diamond LS,Mattern C. Virus of Entamoeba histolyticaⅠIdentification of transmissible virus-like agents[J]. J Virol,1972,9:326~341
[7] Wang AL,Wang CC. The double-stranded RNA in Trichomonoas vaginalis may originate from virus like particles[J]. Proc Natl Acad. 1986,83:7956~7960
[8] Wang AL,Wang CC. Discovery of a specific double-stranded RNA virus in Giardia lamblia[J]. Mol Biochem Parasitol,1986,21:269~276
[9] Revets H,Dekegel D,Deleersnijder W,et al. Identification of virus like particles in Eimeria stiedae[J]. Mol Biochem Parasitol,1989,36:209~216
[10] Johnston RC,Farias NA,Gonzales JC,et al. A putative RNA virus inBabesia bovis[J]. Mol Biochem Parasitol,1991,45:155~158
[11] Tarr PI,Aline RF,Smiley BL,et al. LR1: a candidate RNA virus of Leishmania[J]. Proc Natl Acad Sci USA,1988,85:9572~9575
[12] Wang AL , Wang CC. A linear double 2 stranded RNA in Trichomonas vaginalis[J ] . J Biol Chem , 1985 ,260 :3697~3702
[13] Wang A and Wang C. The double-stranded RNA in Trichomonas vaginalis may originate from virus like particles[J]. Proc.Natl. Acad. Sci. USA, 1986, 83: 7956~7960
[14] Marlene Benchimol, Chang T H, Alderete J F, et al. Trichomonas vaginalis: observation of coexistence of multiple viruses in the same isolate[J]. Fems Microbiol, 2002, 215: 197~201
[15] Marlene Benchimol , Sandra Pacheco Monteiro , et al. Virus in Trichomonas—an ultrastructural study[J]. Parasitology International ,2002, 51:293~298
[16] Champney W S, Curtis S K, Samuels R. Cytopathology and release of an RNA virus from a strain of Trichomonas vaginalis[J].Intern Parasitol, 1995, 5: 1463~1471
[17] Wang AL, Wang CC. Viruses of the protozoa. Annu Rev Microbiol 1991;45:251~63
[18] 赵月平,张西臣,陈丽凤,等.阴道毛滴虫dsRNA病毒部分基因的克隆与序列分析[J].中国寄生虫与寄生虫杂志,2006,24(5): 385~387
[19] A. K ,J. F. Characterization of Double-Stranded RNA Satellites Associated with the Trichomonas vaginalis Virus[J]. Journal of virology, Nov. 1995, 6892–6897
[20] Tai J H and CHUI-FUN I P. The cDNA sequence of Trichomonasvaginalis Virus-T1 Double-Stranded RNA[J]. Virology, 1995, 206: 773~776
[21] Su H M and Tai J H. Genomic organization and sequence conservation in type I Trichomonas vaginalis viruses[J]. Virology, 1996,222 (2): 470~473
[22] Bessarab I N, Liu H W, Ip C F, et al. The complete cDNA sequence of a type II Trichomonas vaginalis virus[J]. Virology, 2000, 267 (2): 350~359
[23] Bessarab I N. and Tai J H. The complete DNA sequence of type Ⅲ Trichomonas vaginalis virus.Unpublished.
[24]. Dailey, D. C., and J. F. Alderete. The phenotypically variable surface protein of Trichomonas vaginalis has a single, tandemly repeated immunodominant epitope. Infect. Immun.1991.59:2083~2088
[25] Esteban R, Fujimura T, and Wickner, R B. Internal and terminal cis-acting sites are necessary for in vitro replication of the LA double-stranded RNA virus of yeast[J]. EMBO, 1989, 8: 947~954
[26] Alderete, J. F., R. Arroyo, D. C. Dailey, J. Engbring, M. A. Khoshnan, and M. W. Lehker. Molecular analysis of Trichomonas vaginalis surface protein repertoires. Mol. Cell Biol. Hum. Dis. Ser. 1992.1:173~202
[27] Alderete, J. F. Alternating phenotypic expression of two classes of Trichomonas vaginalis surface markers. Rev. Infect. Dis. 1988. 10(Suppl. 2) :S408~S412
[28] Alderete, J. F., L. Kasmala, E. Metcalfe, and G. E. Garza. Phenotypic variation and diversity among Trichomonas vaginalis isolates and correlation of phenotype with trichomonal virulence determinants. Infect. Immun. 1986.53:285~293
[29] Khoshnan, A., and J. F. Alderete. Trichomonas vaginalis with a double-stranded RNA virus has upregulated levels of phenotypically variable immunogen mRNA. J. Virol. 1994.68:4035~4038
[30]. Wang, A., C. Wang, and J. F. Alderete. Trichomonas vaginalis phenotypic variation occurs only among trichomonads with the doublestranded RNA virus. J. Exp. Med. 1987.166:142~150
[31] Khoshna Ali and Alderete J F. Multiple Double-Stranded RNA Segments Are Associated with Virus Particles Infecting Trichomonas vaginalis[J]. Virology, 1993, 67(12): 6950~6955
[32] Lauren J S, Pascale M G, Elizabeth M N, et al. Molecular Epidemiology of Metronidazole Resistance in a Population of Trichomonas vaginalis Clinical Isolates[J]. Clin Microbiol, 2000, 38(8): 3004~3009
[33] Boyer JC , Haenni AL. Infectious transcripts and cDNA clone of RNA viruses [J ] . Virology , 1994 , 198(2) : 415 ~426
[34] Johan F,De J,Bart G. Occurrence and transfection of a Giardia virus[J]. Mol Biochem Parasitol,1987,23:85~89
[35] Tai JH,Chang SC,Chou CF,et al.Separation and characterization of two Giardiavirus in the parasitic protozoan Giardia lamblia[J]. Virology,1996,216:124~132
[36] Palese P. RNA virus vectors :where are we and where do we need to go ? [J ] . Proc Natl Acad Sci U S A , 1998 , 95(22) : 12750~12752
[37] TAI et al.The cDNA Sequence of Trichomonas vaginalis Virus-T1 Double-Stranded RNA [J ] .Virology ,1995,206: 773~776
[38] Meulenberg J J M , Bos - de Ruijter J N A , Van De Graaf R , et al.J of Virol , 1998 , 72 :380~387
[39] Prasher DC, Eckenrode V K, Ward WW , et al. Primary structure of the A quorea victoria green-fluo rescent p rotein. Gene, 1992, 111:229
[40] Chalfie M , Tu Y, Euskirchen G, et al. Green -fluo rescent protein as amarker fo r gene exp ression. Science, 1994, 263:802
[41] Lai C,Gouras P,Dio K,et a1.Tracking REP transplants labeled by retroviral gene transfer with gree fluorescent protein Invest[J].Ophthalmol Vis Sei,1999,40:2141~2146
[42] Naylor LH.Reporter gene technology:the future looks bright[J].Bioehem Pharmaeol,1999,58:749~757
[43] Ashwini S K.et al. Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin [J ]. BMC Molecular Biology ,2005, 6:1186~1471
[44] Davis-Hayman SR,Nash TE. Genetic manipulation of Giardia lamblia[J]. Mol Biochem Parasitol,2002,122:1~7
[45] Electric field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells.Mol Cell Biol. 1986,6(2):703~6.
[46] van den Hoff MJB,Moorman A FM,Lamers W H.Eletroporation in intracellular buffer increases cell survival.Nucleic Acids Research,1992,20(11):2902
[47] 周智,张定凤,任红.重组质粒电穿孔转染条件探讨.重庆医科大学学报,1999,24(2):130~132
[48] Shiou-Jeng Ong, Hong-Ming Hsu et al. Multifarious Transcriptional Regulation of Adhesion Protein Gene ap65-1 by a Novel Myb1 Protein in the Protozoan Parasite Trichomonas vaginalis [J].Eukaryot Cell, 2006, 5(2):391~399
[49] Chu-Dang Tsai et al. Characterization of an Iron-responsive Promoter in the Protozoan Pathogen Trichomonas vaginalis [J]. Biol, Chem., 2002 ,277(7) :5153~5162
[2] 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
[3] 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
[4] 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
[5] Mattern CFT,Diamond LS,Daniel TL.Viruses of Entamoeba histolytica. Ⅱ. Morphogenesis of the polyhedral particle (ABRAM2-HK-9)-HB-301 and the filamentous agent (ABRM)2 -HK- 9[J]. J Virol,1972,9:342~358
[6] Diamond LS,Mattern C. Virus of Entamoeba histolyticaⅠIdentification of transmissible virus-like agents[J]. J Virol,1972,9:326~341
[7] Wang AL,Wang CC. The double-stranded RNA in Trichomonoas vaginalis may originate from virus like particles[J]. Proc Natl Acad. 1986,83:7956~7960
[8] Wang AL,Wang CC. Discovery of a specific double-stranded RNA virus in Giardia lamblia[J]. Mol Biochem Parasitol,1986,21:269~276
[9] Revets H,Dekegel D,Deleersnijder W,et al. Identification of virus like particles in Eimeria stiedae[J]. Mol Biochem Parasitol,1989,36:209~216
[10] Johnston RC,Farias NA,Gonzales JC,et al. A putative RNA virus inBabesia bovis[J]. Mol Biochem Parasitol,1991,45:155~158
[11] Tarr PI,Aline RF,Smiley BL,et al. LR1: a candidate RNA virus of Leishmania[J]. Proc Natl Acad Sci USA,1988,85:9572~9575
[12] Wang AL , Wang CC. A linear double 2 stranded RNA in Trichomonas vaginalis[J ] . J Biol Chem , 1985 ,260 :3697~3702
[13] Wang A and Wang C. The double-stranded RNA in Trichomonas vaginalis may originate from virus like particles[J]. Proc.Natl. Acad. Sci. USA, 1986, 83: 7956~7960
[14] Marlene Benchimol, Chang T H, Alderete J F, et al. Trichomonas vaginalis: observation of coexistence of multiple viruses in the same isolate[J]. Fems Microbiol, 2002, 215: 197~201
[15] Marlene Benchimol , Sandra Pacheco Monteiro , et al. Virus in Trichomonas—an ultrastructural study[J]. Parasitology International ,2002, 51:293~298
[16] Champney W S, Curtis S K, Samuels R. Cytopathology and release of an RNA virus from a strain of Trichomonas vaginalis[J].Intern Parasitol, 1995, 5: 1463~1471
[17] Wang AL, Wang CC. Viruses of the protozoa. Annu Rev Microbiol 1991;45:251~63
[18] 赵月平,张西臣,陈丽凤,等.阴道毛滴虫dsRNA病毒部分基因的克隆与序列分析[J].中国寄生虫与寄生虫杂志,2006,24(5): 385~387
[19] A. K ,J. F. Characterization of Double-Stranded RNA Satellites Associated with the Trichomonas vaginalis Virus[J]. Journal of virology, Nov. 1995, 6892–6897
[20] Tai J H and CHUI-FUN I P. The cDNA sequence of Trichomonasvaginalis Virus-T1 Double-Stranded RNA[J]. Virology, 1995, 206: 773~776
[21] Su H M and Tai J H. Genomic organization and sequence conservation in type I Trichomonas vaginalis viruses[J]. Virology, 1996,222 (2): 470~473
[22] Bessarab I N, Liu H W, Ip C F, et al. The complete cDNA sequence of a type II Trichomonas vaginalis virus[J]. Virology, 2000, 267 (2): 350~359
[23] Bessarab I N. and Tai J H. The complete DNA sequence of type Ⅲ Trichomonas vaginalis virus.Unpublished.
[24]. Dailey, D. C., and J. F. Alderete. The phenotypically variable surface protein of Trichomonas vaginalis has a single, tandemly repeated immunodominant epitope. Infect. Immun.1991.59:2083~2088
[25] Esteban R, Fujimura T, and Wickner, R B. Internal and terminal cis-acting sites are necessary for in vitro replication of the LA double-stranded RNA virus of yeast[J]. EMBO, 1989, 8: 947~954
[26] Alderete, J. F., R. Arroyo, D. C. Dailey, J. Engbring, M. A. Khoshnan, and M. W. Lehker. Molecular analysis of Trichomonas vaginalis surface protein repertoires. Mol. Cell Biol. Hum. Dis. Ser. 1992.1:173~202
[27] Alderete, J. F. Alternating phenotypic expression of two classes of Trichomonas vaginalis surface markers. Rev. Infect. Dis. 1988. 10(Suppl. 2) :S408~S412
[28] Alderete, J. F., L. Kasmala, E. Metcalfe, and G. E. Garza. Phenotypic variation and diversity among Trichomonas vaginalis isolates and correlation of phenotype with trichomonal virulence determinants. Infect. Immun. 1986.53:285~293
[29] Khoshnan, A., and J. F. Alderete. Trichomonas vaginalis with a double-stranded RNA virus has upregulated levels of phenotypically variable immunogen mRNA. J. Virol. 1994.68:4035~4038
[30]. Wang, A., C. Wang, and J. F. Alderete. Trichomonas vaginalis phenotypic variation occurs only among trichomonads with the doublestranded RNA virus. J. Exp. Med. 1987.166:142~150
[31] Khoshna Ali and Alderete J F. Multiple Double-Stranded RNA Segments Are Associated with Virus Particles Infecting Trichomonas vaginalis[J]. Virology, 1993, 67(12): 6950~6955
[32] Lauren J S, Pascale M G, Elizabeth M N, et al. Molecular Epidemiology of Metronidazole Resistance in a Population of Trichomonas vaginalis Clinical Isolates[J]. Clin Microbiol, 2000, 38(8): 3004~3009
[33] Boyer JC , Haenni AL. Infectious transcripts and cDNA clone of RNA viruses [J ] . Virology , 1994 , 198(2) : 415 ~426
[34] Johan F,De J,Bart G. Occurrence and transfection of a Giardia virus[J]. Mol Biochem Parasitol,1987,23:85~89
[35] Tai JH,Chang SC,Chou CF,et al.Separation and characterization of two Giardiavirus in the parasitic protozoan Giardia lamblia[J]. Virology,1996,216:124~132
[36] Palese P. RNA virus vectors :where are we and where do we need to go ? [J ] . Proc Natl Acad Sci U S A , 1998 , 95(22) : 12750~12752
[37] TAI et al.The cDNA Sequence of Trichomonas vaginalis Virus-T1 Double-Stranded RNA [J ] .Virology ,1995,206: 773~776
[38] Meulenberg J J M , Bos - de Ruijter J N A , Van De Graaf R , et al.J of Virol , 1998 , 72 :380~387
[39] Prasher DC, Eckenrode V K, Ward WW , et al. Primary structure of the A quorea victoria green-fluo rescent p rotein. Gene, 1992, 111:229
[40] Chalfie M , Tu Y, Euskirchen G, et al. Green -fluo rescent protein as amarker fo r gene exp ression. Science, 1994, 263:802
[41] Lai C,Gouras P,Dio K,et a1.Tracking REP transplants labeled by retroviral gene transfer with gree fluorescent protein Invest[J].Ophthalmol Vis Sei,1999,40:2141~2146
[42] Naylor LH.Reporter gene technology:the future looks bright[J].Bioehem Pharmaeol,1999,58:749~757
[43] Ashwini S K.et al. Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin [J ]. BMC Molecular Biology ,2005, 6:1186~1471
[44] Davis-Hayman SR,Nash TE. Genetic manipulation of Giardia lamblia[J]. Mol Biochem Parasitol,2002,122:1~7
[45] Electric field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells.Mol Cell Biol. 1986,6(2):703~6.
[46] van den Hoff MJB,Moorman A FM,Lamers W H.Eletroporation in intracellular buffer increases cell survival.Nucleic Acids Research,1992,20(11):2902
[47] 周智,张定凤,任红.重组质粒电穿孔转染条件探讨.重庆医科大学学报,1999,24(2):130~132
[48] Shiou-Jeng Ong, Hong-Ming Hsu et al. Multifarious Transcriptional Regulation of Adhesion Protein Gene ap65-1 by a Novel Myb1 Protein in the Protozoan Parasite Trichomonas vaginalis [J].Eukaryot Cell, 2006, 5(2):391~399
[49] Chu-Dang Tsai et al. Characterization of an Iron-responsive Promoter in the Protozoan Pathogen Trichomonas vaginalis [J]. Biol, Chem., 2002 ,277(7) :5153~5162