弓形虫P24基因敲除质粒的构建及缺陷型虫株建立方法的初步研究
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摘要
研究目的:
建立弓形虫P24基因缺陷型虫株,并对缺陷型虫株建立的条件进行探索。
研究方法:
(1)弓形虫P24基因敲除转染质粒pGB/P5-P3的构建
根据TgP24基因序列,设计并合成两对特异引物(P1 to P4),采用PCR技术特异扩增TgP24基因的5'端非翻译区2.5 kb片段(P245'UTR)和3'端非翻译区2.89 kb片段(P24 3'UTR),将其分别亚克隆入pCR2.1-TOPO TA载体。构建质粒P24 5'UTR/TA和P243'UTR/TA;重组质粒P24 5'UTR/TA经KpnⅠ和BglⅡ双酶切后,再将纯化的P24 5'UTR片段亚克隆入转染质粒GRA2/Ble的KpnⅠ和BglⅡ位点,构建重组质粒pGB-P5(GRA2/Ble-P24 5'UTR);重组质粒P24 3'UTR/TA经BamHⅠ和NotⅠ双酶切后,纯化P24 3'UTR片段,再将其定向克隆到重组质粒pGB-P5的BamHⅠ和NotⅠ位点。从而构建TgP24基因敲除转染质粒pGB/P5-P3。
(2)弓形虫P24基因缺陷型虫株筛选条件的建立
采用微孔滤膜(孔径为5.0um)过滤法纯化弓形虫速殖子;比较观察电穿孔条件,如电转染缓冲液,电压,电容,脉冲次数对质粒转染虫株的影响。体外比较观察不同宿主细胞(HFF,3T3和L929)对转染虫株药物筛选的影响;比较观察单独细胞内药物筛选和结合细胞外药物低温处理对虫株筛选的影响(细胞中选择培养10天左右,收集虫体,4℃下福来霉素处理5天,再回复到细胞内用选择培养基培养约7天)。
(3)弓形虫P24基因缺陷型虫株的建立及鉴定分析
应用优化条件的电穿孔方法转染RH株速殖子,以已建立的最佳筛选条件对虫株进行筛选,将虫株继续在细胞中传代培养(12hr),高倍显微镜下,观察纳虫泡的形成并计数,然后大量收集纯化虫株,用RT-PCR及Western-blot两种方法对各组弓形虫P24基因缺陷型虫株的基因及其蛋白表达进行分析,并对虫株的毒力变化进行了初步观察。
研究结果:
(1)弓形虫TgP24基因敲除转染质粒pGB/P5-P3经过PCR筛选、限制性酶切及DNA测序鉴定,证实P24 5'UTR和P24 3'UTR两片段正确插入质粒GRA2/Ble的KpnⅠ和BglⅡ及BamHⅠ和NotⅠ位点,位于药物选择ble基因的上,下游。
(2)显微镜检查发现速殖子纯度可达95%,滤膜过滤虫体后,回收率也可达70%-80%;通过比较观察发现,优化电穿孔条件后的弓形虫存活率得到提高(P＜0.05);采用将细胞内筛选和细胞外药物低温处理相结合的方法对虫株进行筛选较彻底;采用L929成纤维细胞做为转染虫株筛选的宿主细胞其易贴壁生长形成单层,个体较大,细胞传代次数多,生长速度较慢,并且对筛选药物的耐受力为三种细胞中最理想的。
(3)高倍显微镜下计数,12hr左右转染虫株在细胞中纳虫泡数为8个/hp,明显低于RH速殖子形成的纳虫泡数19个/hp(P＜0.05);获得的转染虫株经RT-PCR,Western-blot检测,结果显示L929细胞筛选组虫株的基因及蛋白水平均明显低于HFF和NIH-3T3细胞筛选组虫株:动物毒力试验结果表明,L929细胞筛选组虫株感染小鼠的平均存活时间长于对照组RH虫株。
结论:
(1)成功构建了弓形虫P24基因敲除转染质粒pGB/P5-P3,并且插入的靶基因两端的片段较长,有利于提高同源重组效率。
(2)确定了弓形虫电穿孔技术的应用条件以及TgP24基因敲除质粒转染虫株在不同哺乳动物细胞中的最佳筛选条件(采用细胞内筛选和细胞外药物低温处理相结合的方法,福来霉素浓度为5.0μg/ml-7.5μg/ml,采用L929成纤维细胞做为基因敲除虫株筛选的宿主细胞筛选时间较长,筛选效果好)。
(3)初步获得了弓形虫P24基因缺陷型虫株,为进一步研究弓形虫TgP24基因缺陷型虫株的生物学特性打下了良好的基础。
Objective:
To establish TgP24 gene-knockout strains,and Exploration on the application conditions of screening.Tg P24 gene knockout strains.
Method:
(1)Construction of the targeting plasmid pGB/p5-p3 for Toxoplasma gondii P24 gene(TgP24)knock out.
Accoding to the genomic sequence of TgP24,four oligonudeotides primers(P1 to P4)were designed and synthesized to amplify the 5'end 2.5 kb fragment of untramlated region(UTR)and the 3'end 2.89 kb frarment of UTR of TgP24.The PCR were subdoned into pCR2.1-TOPO TA plasmid to generate the recombinant plasmid P24-5'-UTR/ TA and P24-3'UTR/TA respectively.The purified fragment of P24-5'-UTR from the digestion of P24-5'-UTR/TA with KpnⅠand BglⅡwas inserted into the KpnⅠand BglⅡsites of plasmid GRA2/Ble to generate plasmid GRA2/Ble-P24-5'UTR(designated pGB-P5);The purified P24-3'-UTR fragment from the digestion of plasmid P24-3'-UTR/TA with BamH-Ⅰand NotⅠwas inserted into the BamHⅠand NotⅠsites of plasmid pGB-P5 to generate the targeting plasmid pGB-P5/P24-3' UTR(pGB/P5-P3).
(2)The condition establishment of TgP24 knock-out strain in vitro screening.
Use the millipore filter(aperture 5.0um)filter and purify RH tachyzoites,Observe relatively the impact on plasmid transfected strains of electroporation parameters,such as electroporation transfection buffer, voltage,capacitance,pulse frequency,the size of electric shock Cup The impact of pest.Observe relatively the impact on the transfected strains of different host cells(HFF,3T3 and L929)in vitro on by drug screening;Observe relatively the impact on the transfected strains screened separatly in cell and combine it with low temperature treatment ecto-cells by drug screening(selectively cultured ten years in cells.collected the strains,4℃in extracellular choose five days, retropositioned to the cells cultured in the phleomycin selective medium for seven days).
(3)The establishment、identification and analysis of toxoplasma P24 gene knock-out strains.
Apply the optimized conditions of electroporation method for transfering RH tachyzoites;use the best conditions for screening transferred strains,then serial subcultivation,High-power microscope observe and count the formation of parasitophorous vacuole(PV),large collection and purify Toxoplasma strains,utend RT-PCR and Western-blot two methods analysis about gene and its protein expression of Toxoplasma P24 gene knock-out strains,and research on the toxicity of gene knock-out strains.
Results:
(1)Experimental results showed that the targeting plasmid pGB/P5-P3 was constructed by inserting 2.5 kb and 2.89 kb DNA fragment corresponding to the 5'end and the 3'end UTR of the TgP24 gene into the plasmid GRA2/Ble.The fragment P24-5'UTR was inserted into the poly linker site Kpn-Ⅰ/BglⅡupstream of the phleornycin resistance gene(ble),and the P24-3'UTR frament inserted into BamHⅠ/NotⅠsite down-stream of the same gene.
(2)Microscopic examination revealed the purity of RH tachyzoites can reach 95 percent,the recovery rate of filtered strains also can reach around 70-80 percent.Optimized conditions of electroporation has improved the survival rate of RH tachyzoites(P＜0.05);We relatively observated,the cells will be used within cells,use the method of drug screening on strains in cells and low-temperature treatment ecto-cells will be more thorough;Use L929 fibroblast cell as the host cell for screening,it is easy to form a single Layer,large-volume individual,many passage frequency and slow growth,and the most strong resistant to drug.
(3)High-power microscope observe and count,after about 12hr,the number of the transfected strains PV are 8/hp(per high power microscope),significantly lower than the number of the RH tachyzoites PV are 19/hp(P＜0.05);Then large collection and purification of P24 gene knock-out strains,RT-PCR,Western-blot showed that the gene and protein expression product of strains screened in L929 cells were significantly lower the groups of strains screened in HFF ang NIH-3T3; animal toxicity test results showed that mean survival time of mice attacked strains screened in L929 cells is longest.
Conclusion:
(1)The construction of P24 gene knock-out plasmid pGB/P5-P3 was successfully achieved,and amplified fragment at both ends of the target gene is longer,it will benefit to improve the homologous recombination efficiency.
(2)Ascertain the application conditions of Toxoplasma electroporation technology,and the best screening conditions of TgP24 knockout plasmid transfected into RH strains cultured in different mammalian cell lines(use drug screening in cells and low-temperature treatment ecto-cells,drug selective concentration within 5.0μg/ml-7.5μg/ ml phleomycin,and L929 fibroblast cell have long screening time and good screening effect as selective host cell)
(3)Preliminary obtaining the toxoplasma P24 gene knock-out strains.It has laid a favourable foundation for further research on the biological characteristics of TgP24 knock-out strains.
建立弓形虫P24基因缺陷型虫株,并对缺陷型虫株建立的条件进行探索。
研究方法:
(1)弓形虫P24基因敲除转染质粒pGB/P5-P3的构建
根据TgP24基因序列,设计并合成两对特异引物(P1 to P4),采用PCR技术特异扩增TgP24基因的5'端非翻译区2.5 kb片段(P245'UTR)和3'端非翻译区2.89 kb片段(P24 3'UTR),将其分别亚克隆入pCR2.1-TOPO TA载体。构建质粒P24 5'UTR/TA和P243'UTR/TA;重组质粒P24 5'UTR/TA经KpnⅠ和BglⅡ双酶切后,再将纯化的P24 5'UTR片段亚克隆入转染质粒GRA2/Ble的KpnⅠ和BglⅡ位点,构建重组质粒pGB-P5(GRA2/Ble-P24 5'UTR);重组质粒P24 3'UTR/TA经BamHⅠ和NotⅠ双酶切后,纯化P24 3'UTR片段,再将其定向克隆到重组质粒pGB-P5的BamHⅠ和NotⅠ位点。从而构建TgP24基因敲除转染质粒pGB/P5-P3。
(2)弓形虫P24基因缺陷型虫株筛选条件的建立
采用微孔滤膜(孔径为5.0um)过滤法纯化弓形虫速殖子;比较观察电穿孔条件,如电转染缓冲液,电压,电容,脉冲次数对质粒转染虫株的影响。体外比较观察不同宿主细胞(HFF,3T3和L929)对转染虫株药物筛选的影响;比较观察单独细胞内药物筛选和结合细胞外药物低温处理对虫株筛选的影响(细胞中选择培养10天左右,收集虫体,4℃下福来霉素处理5天,再回复到细胞内用选择培养基培养约7天)。
(3)弓形虫P24基因缺陷型虫株的建立及鉴定分析
应用优化条件的电穿孔方法转染RH株速殖子,以已建立的最佳筛选条件对虫株进行筛选,将虫株继续在细胞中传代培养(12hr),高倍显微镜下,观察纳虫泡的形成并计数,然后大量收集纯化虫株,用RT-PCR及Western-blot两种方法对各组弓形虫P24基因缺陷型虫株的基因及其蛋白表达进行分析,并对虫株的毒力变化进行了初步观察。
研究结果:
(1)弓形虫TgP24基因敲除转染质粒pGB/P5-P3经过PCR筛选、限制性酶切及DNA测序鉴定,证实P24 5'UTR和P24 3'UTR两片段正确插入质粒GRA2/Ble的KpnⅠ和BglⅡ及BamHⅠ和NotⅠ位点,位于药物选择ble基因的上,下游。
(2)显微镜检查发现速殖子纯度可达95%,滤膜过滤虫体后,回收率也可达70%-80%;通过比较观察发现,优化电穿孔条件后的弓形虫存活率得到提高(P＜0.05);采用将细胞内筛选和细胞外药物低温处理相结合的方法对虫株进行筛选较彻底;采用L929成纤维细胞做为转染虫株筛选的宿主细胞其易贴壁生长形成单层,个体较大,细胞传代次数多,生长速度较慢,并且对筛选药物的耐受力为三种细胞中最理想的。
(3)高倍显微镜下计数,12hr左右转染虫株在细胞中纳虫泡数为8个/hp,明显低于RH速殖子形成的纳虫泡数19个/hp(P＜0.05);获得的转染虫株经RT-PCR,Western-blot检测,结果显示L929细胞筛选组虫株的基因及蛋白水平均明显低于HFF和NIH-3T3细胞筛选组虫株:动物毒力试验结果表明,L929细胞筛选组虫株感染小鼠的平均存活时间长于对照组RH虫株。
结论:
(1)成功构建了弓形虫P24基因敲除转染质粒pGB/P5-P3,并且插入的靶基因两端的片段较长,有利于提高同源重组效率。
(2)确定了弓形虫电穿孔技术的应用条件以及TgP24基因敲除质粒转染虫株在不同哺乳动物细胞中的最佳筛选条件(采用细胞内筛选和细胞外药物低温处理相结合的方法,福来霉素浓度为5.0μg/ml-7.5μg/ml,采用L929成纤维细胞做为基因敲除虫株筛选的宿主细胞筛选时间较长,筛选效果好)。
(3)初步获得了弓形虫P24基因缺陷型虫株,为进一步研究弓形虫TgP24基因缺陷型虫株的生物学特性打下了良好的基础。
Objective:
To establish TgP24 gene-knockout strains,and Exploration on the application conditions of screening.Tg P24 gene knockout strains.
Method:
(1)Construction of the targeting plasmid pGB/p5-p3 for Toxoplasma gondii P24 gene(TgP24)knock out.
Accoding to the genomic sequence of TgP24,four oligonudeotides primers(P1 to P4)were designed and synthesized to amplify the 5'end 2.5 kb fragment of untramlated region(UTR)and the 3'end 2.89 kb frarment of UTR of TgP24.The PCR were subdoned into pCR2.1-TOPO TA plasmid to generate the recombinant plasmid P24-5'-UTR/ TA and P24-3'UTR/TA respectively.The purified fragment of P24-5'-UTR from the digestion of P24-5'-UTR/TA with KpnⅠand BglⅡwas inserted into the KpnⅠand BglⅡsites of plasmid GRA2/Ble to generate plasmid GRA2/Ble-P24-5'UTR(designated pGB-P5);The purified P24-3'-UTR fragment from the digestion of plasmid P24-3'-UTR/TA with BamH-Ⅰand NotⅠwas inserted into the BamHⅠand NotⅠsites of plasmid pGB-P5 to generate the targeting plasmid pGB-P5/P24-3' UTR(pGB/P5-P3).
(2)The condition establishment of TgP24 knock-out strain in vitro screening.
Use the millipore filter(aperture 5.0um)filter and purify RH tachyzoites,Observe relatively the impact on plasmid transfected strains of electroporation parameters,such as electroporation transfection buffer, voltage,capacitance,pulse frequency,the size of electric shock Cup The impact of pest.Observe relatively the impact on the transfected strains of different host cells(HFF,3T3 and L929)in vitro on by drug screening;Observe relatively the impact on the transfected strains screened separatly in cell and combine it with low temperature treatment ecto-cells by drug screening(selectively cultured ten years in cells.collected the strains,4℃in extracellular choose five days, retropositioned to the cells cultured in the phleomycin selective medium for seven days).
(3)The establishment、identification and analysis of toxoplasma P24 gene knock-out strains.
Apply the optimized conditions of electroporation method for transfering RH tachyzoites;use the best conditions for screening transferred strains,then serial subcultivation,High-power microscope observe and count the formation of parasitophorous vacuole(PV),large collection and purify Toxoplasma strains,utend RT-PCR and Western-blot two methods analysis about gene and its protein expression of Toxoplasma P24 gene knock-out strains,and research on the toxicity of gene knock-out strains.
Results:
(1)Experimental results showed that the targeting plasmid pGB/P5-P3 was constructed by inserting 2.5 kb and 2.89 kb DNA fragment corresponding to the 5'end and the 3'end UTR of the TgP24 gene into the plasmid GRA2/Ble.The fragment P24-5'UTR was inserted into the poly linker site Kpn-Ⅰ/BglⅡupstream of the phleornycin resistance gene(ble),and the P24-3'UTR frament inserted into BamHⅠ/NotⅠsite down-stream of the same gene.
(2)Microscopic examination revealed the purity of RH tachyzoites can reach 95 percent,the recovery rate of filtered strains also can reach around 70-80 percent.Optimized conditions of electroporation has improved the survival rate of RH tachyzoites(P＜0.05);We relatively observated,the cells will be used within cells,use the method of drug screening on strains in cells and low-temperature treatment ecto-cells will be more thorough;Use L929 fibroblast cell as the host cell for screening,it is easy to form a single Layer,large-volume individual,many passage frequency and slow growth,and the most strong resistant to drug.
(3)High-power microscope observe and count,after about 12hr,the number of the transfected strains PV are 8/hp(per high power microscope),significantly lower than the number of the RH tachyzoites PV are 19/hp(P＜0.05);Then large collection and purification of P24 gene knock-out strains,RT-PCR,Western-blot showed that the gene and protein expression product of strains screened in L929 cells were significantly lower the groups of strains screened in HFF ang NIH-3T3; animal toxicity test results showed that mean survival time of mice attacked strains screened in L929 cells is longest.
Conclusion:
(1)The construction of P24 gene knock-out plasmid pGB/P5-P3 was successfully achieved,and amplified fragment at both ends of the target gene is longer,it will benefit to improve the homologous recombination efficiency.
(2)Ascertain the application conditions of Toxoplasma electroporation technology,and the best screening conditions of TgP24 knockout plasmid transfected into RH strains cultured in different mammalian cell lines(use drug screening in cells and low-temperature treatment ecto-cells,drug selective concentration within 5.0μg/ml-7.5μg/ ml phleomycin,and L929 fibroblast cell have long screening time and good screening effect as selective host cell)
(3)Preliminary obtaining the toxoplasma P24 gene knock-out strains.It has laid a favourable foundation for further research on the biological characteristics of TgP24 knock-out strains.
引文
[1]Sibley LD,Niesman IR,Pamley SF,et al.Regulated secretion of multi-lamellar vesicles leads to forrmation of a tubulo-vesicular network in host-cell vacuol occupied by Toxoplasrtm gondii[J].J Cell Sci,1995,108:1669-1677.
[2]Sibley LD,Boothroyd JC.Construction of a molecular karyotype for Toxoplasma gondii[J].Mol Biochem Parasitol.1992,51:291-300.
[3]Lecordier L,Fourmaux MP,Mereier C,et al.Enzyme-linked immunosorbent assays using the recombinant dense granule antigens GRA6 and GRA1 of Toxoplasma gondii for detection of immunoglobulin G antibodies[J].Clin Diagn Lab Imnmnol.2000,7:607-611.
[4]Cesbron-Delauw MF,Guy B,Torpier G,et aI.Molecular characterization of a 23kilodalton major antigen secreted by Toxoplasma gondii[J].Proc Natl Acad Sci USA,1989,86:7537-7541.
[5]Scorza T,D'Souza S.Laloup M,et al.A GRA1 DNA vaccine primes cytolytic CD8~ T cells to control acute Toxoplasma gondii infection[J].Infect Immun,2003,71(1):309-316.es
[6]舒衡平,蒋立平,罗树红弓形虫P24基因(TgP24)敲除转染质粒pGB/P5-P3的构建.[J].中国人兽共患病杂志,2004,20(11):926-929.
[7]Smithies O,Gregg R G,Boggs SS,et al.Isertion of DNA sequences into the human chromosomal betaglobin locus by homologous recobination[J].Nature,1985,317(6034):230-234.
[8]Mercier C,Howe DK,Mordue D,Lingnau M,Sibley LD.Targeted Disruption of the GRA2 Locus in Toxoplasma gondii Decreases Acute Virulence in Mice[J]..Infect Immun.1998,66(9):4176-4182.
[9]Mercier C,Rauscher B,Lecordier L,et al.Lack of the dense granule protein GRA5does not affect the development of Toxoplasma tachyzoites[J].Mol Biochem parasitol,2001,116(2001):247-251.
[10]Cerede O,Dubremetz JF,Soete M,Deslee D,Vial H,.Bout D,Lebrun M.Synergistic role of micronemal proteins in Toxoplasma gondii virulence[J].J Exp Med.2005 Feb 7;201(3):453-463.Epub 2005 Jan 31.
[11]詹希美.人体寄生虫学[M].北京:人民卫生出版社,2005,93-94
[12]Evans R,Chatterton JM,Ashburn D,et al.Cell-culture system for continuoous prodoction of Toxoplasma gondii tachyzoites[J].Eur J Clin Microbiol Infect Dis,1999,18(12):879-884.
[13]许丽芳,杨秋林,张愉快,等.用包皮成纤维细胞培养弓形虫速殖子的研究[J].中国寄生虫病防治杂志.2004.17(5):268-270.
[14]王文实,杨秀珍,杨树森.三株弓形虫速殖子的体外培养研究[J].中国人兽共患病杂志,1999,15(1):6-8.
[15]李立伟,邵浙新,严杰.刚地弓形虫对HUVEC、Vero和J774A.1、THPl 侵入的比较研究[J]。中国人兽共患病学报.2006,22(7):597-600.
[16]Donald RG,Roos DS.Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii [J].Mol Biochem Parasitol.1994,63(2):243-253.
[17]Brecht S,Erdhart H,Soete M,Soldati D.Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre[J].Gene,1999,234(2):239-247.
[18]Dzierszinski F,Mortuaire M,et al.Targeted disruption of the glycosylp-hosphatidylinositol -anchored surface antigen SAG3 gene in Toxoplasma gondii decreases host cell adhesion and drastically reduces virulence in mice[J].Mol Microbiol,2000,37(3):574-582.
[19]Di Cristina M,Spaceapelo R,et al.Two conserved amino acid motifs mediate protein targeting to the micronemes of the apicomplexan parasite Toxoplasma gondii[J].Mol Cell Biol,2000,20(19):7332-7341.
[20]Robert G.K.Donald,David S.Roos.Gene knock-outs and allelic replacements in Toxoplasma gondii:HXGPRT as a selectable marker for hit-and-run mutagenesis[J].Molecular and Biochemical Parasitology,91(1998)295-305.
[21]shu-hong luo,Felix A.Ruiz,Silvia N.J.Moreno.The acidocalcisome Ca~(2+)-ATPase(TgA1)of Toxoplasma gondii is required for polyphosphate storage,intracelluer calcium homeostasis and virulence[J].Molecular Microbi-ology,2005,55(4):1034-1045.
[22]MJ van den Hoff,Moorman AF,Lames WH.Electroporation in intracellular buffer increases cell survival.Nucleic Acids Res,1992,20(11):2920.
[23]RP Dempster.Toxoplasma gongii:purification of zoites from peritoneal exudates by eight methods.Exp Parasitol.1984.57(2):195-207.
[24]R Robert,Mauras G,Senet JM,et al.Purification of Toxoplasma gondii suspensionsfrom mice peritoneal exudate[J].Biomed Pharmacother.1982. 36(2):98-100.
[25]郑焕钦.一种新的弓形虫速殖子纯化方法[J].中国寄生虫病防治杂志,1993,6(3):146.
[26]刘佩酶,吴增强,武苏平.弓形虫速殖子纯化方法的比较[J].天津医科大学学报,1996,2(1):59-60.
[27]沈继龙.组织内弓形虫病原体的分离纯化[J].中国人兽共患病杂志,1991,7(5):2-4.
[28]S Tomavo,Dubremetz JF,Schwarz RT.Biosynthesis of glycolipid precursors for glycosylphosphatidylinositol membrane anchors in a Toxoplasma gondii cell-free system[J].J Biol Chem,1992,267(30):21446-214458.
[29]M Soete,D Camus,J.F Dubremetz.Experimental induction of bradyzoite -specific antigen expression and cyst formation by the RH strain of ToxopIasma gondii in vitro[J].Exp.Parasitol,1994,78(4):361-370.
[30]D Soldati,Boothroyd JC.Transient.transfection and expression in the obligate intracellular parasite Toxoplasma gondii.Science.1993,260(5106):349-352.
[31](美)萨姆布鲁克(sambrook,J)等著;黄培堂等译.分子克隆实验指南.第三版.北京:科学出版社,2002.
[32]Ute Schepers.实用RNAi技术-线虫、果蝇和哺乳动物细胞基因沉默的基本原理与方法[M].北京:人民卫生出版社.第一版.2006,58-78.
[33]MS Eaton,Weiss LM,Kim K.Cyclic nucleotide kinases and tachyzoite transtition in Toxoplasma gondii[J].Int J Parasitol.2006.36(1):107-114.
[34]C Toursel,Dzierszinski F,Bernigaud A,et al.Molecular cloning,organellar targeting and developmental expression of mitochondrial chaperone HSP60 in Toxoplasma gondii[J].Mol Biochem Parasitol.2000,111(2):319-332.
[35]RG Donald,Carter D,Ullman B,et al.Insertional tagging,cloning,and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferse gene.Use as a selectable marker for stable transformation[J].J Biol Chem.1996,271(24):14010-14019.
[36]W Bohne,Roos DS.Stage-specifiC expression of a slectable marker in Toxoplama gondii permits selective inhibition of either tachyzoites or bradyzoites[J].Mol Biochem Parasitol.1997,88(1-2):115-126.
[37]RG Donald,Roos DS.Gene knock-outs and allelic replacements in Toxoplasma gondii:HXGPRT as a selectable marker for hit-and-run mutagensis[J].Mol Biochem Parasitol.1998,91(2):295-305.
[38]B Striepen,Soldati D,Garcia-Reguet N,et et al.Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii[J].Mol Biochem Parasitol.2001,113(1):45-53.
[39]PJ Bradley,Li N,Boothroyald JC.A GFP-based motif-trap reveals anovel mechanism off targeting for the Toxoplama gondii ROP4 protein[J].Mol Biochem Parasitol.2004,137(1):111-120.
[40]F Dzierszinski,Nishi M,Ouko L,et al.Dynamics of Toxoplasma gondii differentiation[J].Eukaryot Cell.2004,3(4):992-1003.
[1]Tybulewicz VL.Animal model of Gaucher's disease from targeted disruption of the mouse glucocerebrosidase gene.Nature.1992,357(6377):407-410.
[2]Thompson S.Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells.Cell,1989,27;56(2):313-321.
[3]Smithies O,Gregg R G,Boggs SS,et al.Isertion of DNA sequences into the human chromosomal betaglobin locus by homologous recobination.Nature,1985,317(6034):230-234.
[4]Bradley A,et al.Formation of germ-line chimeras from embro derived teratocarcinoma cell.Nature,1984,309:225-256.
[5]Galli-Taliadoros LA,Sedgwick JD,Wood SA,Komer H.Gene knock-out technology:a methodological overview for the interested novice.J Immunol Methods.1995,181(1):1-15.
[6]Mansour S L,Thomas K R,Capccchi M R.Disruption of the protooncogene int-2 in mouse embryoderiver stem ceils.Nature,1988,336(6197):348-352.
[7]Gu H,Marth J d,Orban P C..Deletion of a DNA ploymerase gene segment in T cells using cell type-specific targeting.science,1994,265:103-106.
[8]Yagi T,Tokunaga T,Furuta Y,Nada S,Aizawa S,et al.A novel ES cell line TT2 with high germline-differentiating potency.Analyt Biochem,1993,214(1):70-76
[9]Bautista D,Shulman MJ.A hit-and-run system for introducing mutations into the Ig H chain locus of hybridoma cells by homologous recombination.J Immunol.1993,151(4):1950-8.
[I0]Hasty P,Ramirez-Solis R,Krumlauf R,et al.Introduction of a subtle mutatio into the Hox-2.6 locus in embryonic stem cells.Nature,1991,350:243-246
[11]Askew G R,Doetschman T,Lingrel J B.Site-directed point mutations i embryonic stem cells:a gene-targeting tag-andexchange strategy.Mol Cell Bio 1993,13:4115-4124
[12]Yang X,Zhang Z,Huang C.The strategies of gene targeting in mouse embryoni stem cells.Progress in Biochemistry and Biophysics,1997,24:104-108
[13]Richard C.Moore,Nicola J.et al.Double Replacement Gene Targeting for the Production of a Series of Mouse Strains with Different Prion Protein Gene Alterations.Bio/Technology.13,999-1004(1995)
[14]Fire A,Xu S,Montgrnery M K,et al.Potent and specific geneticintefference by double-strandedRNAinCaenorhabditiselegans.Nature,1998,391:806-811.
[15]Zamore PD,Tuschl T,Sharp PA,et al.RNAi:double stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Cel,2000,101(1):25-33.
[16]Kasschau KD,et al.counterdefensive strate of plant viruses:uppression of posttranscriptional gene silencing.Cell,1998,95(4):461-470.
[17]吴红.基因敲出动物模型的建立.实验动物科学与管理[J].2002,19(1):32-35.
[18]Donehower L,Harvey A M,Slagle B,et al.Mice deficient for p53 are devolopmentally normal but susceptibla to spontaneous tumours.Nature,1992,356(6366):215-221.
[19]程震,黄长晖,等。基因打靶。生命化学,1997,17(2):34-35.
[20]Menard R,Sultan AA,et al.Circumsporozoite protein is required for development of malaria sporozoites inmosquitoes.Nature,1997,385(6614):336-340.
[21]Sultan AA,Thathy V,et al.TRAP is necessary for gliding motility and infectivity of plasmodium sporozoites.Cell.1997 Aug 8;90(3):511-522.
[22] Santrich C, Moore L, et al. A motility function for the paraflagellar rod of Leishmania parasites revealed by PFR-2 gene knockouts.Mol Biochem Parasitol. 1997 Dec 1;90(1):95-109.
[23] PJ Bradley,Li N,Boothroyd JC.A GFP-based motif-trap reveals a novel mechanism of targeting for the Toxoplasma ROP4 protein.Mol Biochem Parasitol.2004.137(1): 111-120.
[24] Al-Anouti F, Ananvoranich S. Comparative analysis of antisense RNA,double-stranded RNA, and delta ribozyme-mediated gene regulation in Toxoplasma gondii. Antisense Nucl Acid Drug.2002,12: 275-281.
[25] Brian Adams, Alia Musiyenko, Rajinder Kumar, et al.A Novel Class of Dual-family ImmunopHilins. J. Biol. Chem., 2005; 280 (26): 24308-24314.
[26] Wang cc.Validating targets for antiparasite chemotherapy. 1997; 114 Suppl:S31-44.
[27] Hunger Gl.et al. Deletion of the genes for the paraflagellar rod protein PFR-A in Trypanosoma brucei is probably lethal. Mol Biochem Parasitol, 1997; 90(1): 347-351.
[28] Sibley LD,et al.Stable DNA transformation in the obligate intracellualar parasite Toxoplasma gondii by complemention of tryptophan auxotrophy,Proc Natl Acad Sci USA,1994,91:5508.
[29] von der Weid, T., Honarvar, N. & Langhorne, J. Gene-targeted mice lacking B cells are unable to eliminate a blood stage malaria infection. J. Immunol. 1996,156,2510-2516
[30] Mercier C, Howe DK, Mordue D, Lingnau M, Sibley LD.Targeted Disruption of the GRA2 Locus in Toxoplasma gondii Decreases Acute Virulence in Mice Infect Immun. 1998 ,66(9):4176-4182.
[31] Cérède O, Dubremetz JF, So(e|^)te M,etal. Synergistic role of micronemal proteins in Toxoplasma gondii virulence. J Exp Med. 2005,201(3):453-63. Epub 2005 Jan 31.
[32] Titus RG,Gueiros-Filho-FJ,de-Freitas-LA et al. Development of a safe live Leishmania vaccine line by gene replacement. Proc Natl Acad Sci USA, 1995,92(22): 10267-10271
[33] Robert Ménard. Knockout malaria vaccine? Nature , 2005,433, 113-114.
[34] Mueller, A.-K., Labaied, M., Kappe, S. H. I. & Matuschewski, K. Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature,433,164-167 (2005).
[35] Cowman AF,McFadden GI,et al.Dissecting apicoplast targeting in the malaria parasite Plasmodium falciparum. Science,2003,299(5607):705-708.
[36] Engers HD et al. Progress on vaccines against parasites.Dev Biol Stand. 1996,87:73-84. Review.
[2]Sibley LD,Boothroyd JC.Construction of a molecular karyotype for Toxoplasma gondii[J].Mol Biochem Parasitol.1992,51:291-300.
[3]Lecordier L,Fourmaux MP,Mereier C,et al.Enzyme-linked immunosorbent assays using the recombinant dense granule antigens GRA6 and GRA1 of Toxoplasma gondii for detection of immunoglobulin G antibodies[J].Clin Diagn Lab Imnmnol.2000,7:607-611.
[4]Cesbron-Delauw MF,Guy B,Torpier G,et aI.Molecular characterization of a 23kilodalton major antigen secreted by Toxoplasma gondii[J].Proc Natl Acad Sci USA,1989,86:7537-7541.
[5]Scorza T,D'Souza S.Laloup M,et al.A GRA1 DNA vaccine primes cytolytic CD8~ T cells to control acute Toxoplasma gondii infection[J].Infect Immun,2003,71(1):309-316.es
[6]舒衡平,蒋立平,罗树红弓形虫P24基因(TgP24)敲除转染质粒pGB/P5-P3的构建.[J].中国人兽共患病杂志,2004,20(11):926-929.
[7]Smithies O,Gregg R G,Boggs SS,et al.Isertion of DNA sequences into the human chromosomal betaglobin locus by homologous recobination[J].Nature,1985,317(6034):230-234.
[8]Mercier C,Howe DK,Mordue D,Lingnau M,Sibley LD.Targeted Disruption of the GRA2 Locus in Toxoplasma gondii Decreases Acute Virulence in Mice[J]..Infect Immun.1998,66(9):4176-4182.
[9]Mercier C,Rauscher B,Lecordier L,et al.Lack of the dense granule protein GRA5does not affect the development of Toxoplasma tachyzoites[J].Mol Biochem parasitol,2001,116(2001):247-251.
[10]Cerede O,Dubremetz JF,Soete M,Deslee D,Vial H,.Bout D,Lebrun M.Synergistic role of micronemal proteins in Toxoplasma gondii virulence[J].J Exp Med.2005 Feb 7;201(3):453-463.Epub 2005 Jan 31.
[11]詹希美.人体寄生虫学[M].北京:人民卫生出版社,2005,93-94
[12]Evans R,Chatterton JM,Ashburn D,et al.Cell-culture system for continuoous prodoction of Toxoplasma gondii tachyzoites[J].Eur J Clin Microbiol Infect Dis,1999,18(12):879-884.
[13]许丽芳,杨秋林,张愉快,等.用包皮成纤维细胞培养弓形虫速殖子的研究[J].中国寄生虫病防治杂志.2004.17(5):268-270.
[14]王文实,杨秀珍,杨树森.三株弓形虫速殖子的体外培养研究[J].中国人兽共患病杂志,1999,15(1):6-8.
[15]李立伟,邵浙新,严杰.刚地弓形虫对HUVEC、Vero和J774A.1、THPl 侵入的比较研究[J]。中国人兽共患病学报.2006,22(7):597-600.
[16]Donald RG,Roos DS.Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii [J].Mol Biochem Parasitol.1994,63(2):243-253.
[17]Brecht S,Erdhart H,Soete M,Soldati D.Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre[J].Gene,1999,234(2):239-247.
[18]Dzierszinski F,Mortuaire M,et al.Targeted disruption of the glycosylp-hosphatidylinositol -anchored surface antigen SAG3 gene in Toxoplasma gondii decreases host cell adhesion and drastically reduces virulence in mice[J].Mol Microbiol,2000,37(3):574-582.
[19]Di Cristina M,Spaceapelo R,et al.Two conserved amino acid motifs mediate protein targeting to the micronemes of the apicomplexan parasite Toxoplasma gondii[J].Mol Cell Biol,2000,20(19):7332-7341.
[20]Robert G.K.Donald,David S.Roos.Gene knock-outs and allelic replacements in Toxoplasma gondii:HXGPRT as a selectable marker for hit-and-run mutagenesis[J].Molecular and Biochemical Parasitology,91(1998)295-305.
[21]shu-hong luo,Felix A.Ruiz,Silvia N.J.Moreno.The acidocalcisome Ca~(2+)-ATPase(TgA1)of Toxoplasma gondii is required for polyphosphate storage,intracelluer calcium homeostasis and virulence[J].Molecular Microbi-ology,2005,55(4):1034-1045.
[22]MJ van den Hoff,Moorman AF,Lames WH.Electroporation in intracellular buffer increases cell survival.Nucleic Acids Res,1992,20(11):2920.
[23]RP Dempster.Toxoplasma gongii:purification of zoites from peritoneal exudates by eight methods.Exp Parasitol.1984.57(2):195-207.
[24]R Robert,Mauras G,Senet JM,et al.Purification of Toxoplasma gondii suspensionsfrom mice peritoneal exudate[J].Biomed Pharmacother.1982. 36(2):98-100.
[25]郑焕钦.一种新的弓形虫速殖子纯化方法[J].中国寄生虫病防治杂志,1993,6(3):146.
[26]刘佩酶,吴增强,武苏平.弓形虫速殖子纯化方法的比较[J].天津医科大学学报,1996,2(1):59-60.
[27]沈继龙.组织内弓形虫病原体的分离纯化[J].中国人兽共患病杂志,1991,7(5):2-4.
[28]S Tomavo,Dubremetz JF,Schwarz RT.Biosynthesis of glycolipid precursors for glycosylphosphatidylinositol membrane anchors in a Toxoplasma gondii cell-free system[J].J Biol Chem,1992,267(30):21446-214458.
[29]M Soete,D Camus,J.F Dubremetz.Experimental induction of bradyzoite -specific antigen expression and cyst formation by the RH strain of ToxopIasma gondii in vitro[J].Exp.Parasitol,1994,78(4):361-370.
[30]D Soldati,Boothroyd JC.Transient.transfection and expression in the obligate intracellular parasite Toxoplasma gondii.Science.1993,260(5106):349-352.
[31](美)萨姆布鲁克(sambrook,J)等著;黄培堂等译.分子克隆实验指南.第三版.北京:科学出版社,2002.
[32]Ute Schepers.实用RNAi技术-线虫、果蝇和哺乳动物细胞基因沉默的基本原理与方法[M].北京:人民卫生出版社.第一版.2006,58-78.
[33]MS Eaton,Weiss LM,Kim K.Cyclic nucleotide kinases and tachyzoite transtition in Toxoplasma gondii[J].Int J Parasitol.2006.36(1):107-114.
[34]C Toursel,Dzierszinski F,Bernigaud A,et al.Molecular cloning,organellar targeting and developmental expression of mitochondrial chaperone HSP60 in Toxoplasma gondii[J].Mol Biochem Parasitol.2000,111(2):319-332.
[35]RG Donald,Carter D,Ullman B,et al.Insertional tagging,cloning,and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferse gene.Use as a selectable marker for stable transformation[J].J Biol Chem.1996,271(24):14010-14019.
[36]W Bohne,Roos DS.Stage-specifiC expression of a slectable marker in Toxoplama gondii permits selective inhibition of either tachyzoites or bradyzoites[J].Mol Biochem Parasitol.1997,88(1-2):115-126.
[37]RG Donald,Roos DS.Gene knock-outs and allelic replacements in Toxoplasma gondii:HXGPRT as a selectable marker for hit-and-run mutagensis[J].Mol Biochem Parasitol.1998,91(2):295-305.
[38]B Striepen,Soldati D,Garcia-Reguet N,et et al.Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii[J].Mol Biochem Parasitol.2001,113(1):45-53.
[39]PJ Bradley,Li N,Boothroyald JC.A GFP-based motif-trap reveals anovel mechanism off targeting for the Toxoplama gondii ROP4 protein[J].Mol Biochem Parasitol.2004,137(1):111-120.
[40]F Dzierszinski,Nishi M,Ouko L,et al.Dynamics of Toxoplasma gondii differentiation[J].Eukaryot Cell.2004,3(4):992-1003.
[1]Tybulewicz VL.Animal model of Gaucher's disease from targeted disruption of the mouse glucocerebrosidase gene.Nature.1992,357(6377):407-410.
[2]Thompson S.Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells.Cell,1989,27;56(2):313-321.
[3]Smithies O,Gregg R G,Boggs SS,et al.Isertion of DNA sequences into the human chromosomal betaglobin locus by homologous recobination.Nature,1985,317(6034):230-234.
[4]Bradley A,et al.Formation of germ-line chimeras from embro derived teratocarcinoma cell.Nature,1984,309:225-256.
[5]Galli-Taliadoros LA,Sedgwick JD,Wood SA,Komer H.Gene knock-out technology:a methodological overview for the interested novice.J Immunol Methods.1995,181(1):1-15.
[6]Mansour S L,Thomas K R,Capccchi M R.Disruption of the protooncogene int-2 in mouse embryoderiver stem ceils.Nature,1988,336(6197):348-352.
[7]Gu H,Marth J d,Orban P C..Deletion of a DNA ploymerase gene segment in T cells using cell type-specific targeting.science,1994,265:103-106.
[8]Yagi T,Tokunaga T,Furuta Y,Nada S,Aizawa S,et al.A novel ES cell line TT2 with high germline-differentiating potency.Analyt Biochem,1993,214(1):70-76
[9]Bautista D,Shulman MJ.A hit-and-run system for introducing mutations into the Ig H chain locus of hybridoma cells by homologous recombination.J Immunol.1993,151(4):1950-8.
[I0]Hasty P,Ramirez-Solis R,Krumlauf R,et al.Introduction of a subtle mutatio into the Hox-2.6 locus in embryonic stem cells.Nature,1991,350:243-246
[11]Askew G R,Doetschman T,Lingrel J B.Site-directed point mutations i embryonic stem cells:a gene-targeting tag-andexchange strategy.Mol Cell Bio 1993,13:4115-4124
[12]Yang X,Zhang Z,Huang C.The strategies of gene targeting in mouse embryoni stem cells.Progress in Biochemistry and Biophysics,1997,24:104-108
[13]Richard C.Moore,Nicola J.et al.Double Replacement Gene Targeting for the Production of a Series of Mouse Strains with Different Prion Protein Gene Alterations.Bio/Technology.13,999-1004(1995)
[14]Fire A,Xu S,Montgrnery M K,et al.Potent and specific geneticintefference by double-strandedRNAinCaenorhabditiselegans.Nature,1998,391:806-811.
[15]Zamore PD,Tuschl T,Sharp PA,et al.RNAi:double stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Cel,2000,101(1):25-33.
[16]Kasschau KD,et al.counterdefensive strate of plant viruses:uppression of posttranscriptional gene silencing.Cell,1998,95(4):461-470.
[17]吴红.基因敲出动物模型的建立.实验动物科学与管理[J].2002,19(1):32-35.
[18]Donehower L,Harvey A M,Slagle B,et al.Mice deficient for p53 are devolopmentally normal but susceptibla to spontaneous tumours.Nature,1992,356(6366):215-221.
[19]程震,黄长晖,等。基因打靶。生命化学,1997,17(2):34-35.
[20]Menard R,Sultan AA,et al.Circumsporozoite protein is required for development of malaria sporozoites inmosquitoes.Nature,1997,385(6614):336-340.
[21]Sultan AA,Thathy V,et al.TRAP is necessary for gliding motility and infectivity of plasmodium sporozoites.Cell.1997 Aug 8;90(3):511-522.
[22] Santrich C, Moore L, et al. A motility function for the paraflagellar rod of Leishmania parasites revealed by PFR-2 gene knockouts.Mol Biochem Parasitol. 1997 Dec 1;90(1):95-109.
[23] PJ Bradley,Li N,Boothroyd JC.A GFP-based motif-trap reveals a novel mechanism of targeting for the Toxoplasma ROP4 protein.Mol Biochem Parasitol.2004.137(1): 111-120.
[24] Al-Anouti F, Ananvoranich S. Comparative analysis of antisense RNA,double-stranded RNA, and delta ribozyme-mediated gene regulation in Toxoplasma gondii. Antisense Nucl Acid Drug.2002,12: 275-281.
[25] Brian Adams, Alia Musiyenko, Rajinder Kumar, et al.A Novel Class of Dual-family ImmunopHilins. J. Biol. Chem., 2005; 280 (26): 24308-24314.
[26] Wang cc.Validating targets for antiparasite chemotherapy. 1997; 114 Suppl:S31-44.
[27] Hunger Gl.et al. Deletion of the genes for the paraflagellar rod protein PFR-A in Trypanosoma brucei is probably lethal. Mol Biochem Parasitol, 1997; 90(1): 347-351.
[28] Sibley LD,et al.Stable DNA transformation in the obligate intracellualar parasite Toxoplasma gondii by complemention of tryptophan auxotrophy,Proc Natl Acad Sci USA,1994,91:5508.
[29] von der Weid, T., Honarvar, N. & Langhorne, J. Gene-targeted mice lacking B cells are unable to eliminate a blood stage malaria infection. J. Immunol. 1996,156,2510-2516
[30] Mercier C, Howe DK, Mordue D, Lingnau M, Sibley LD.Targeted Disruption of the GRA2 Locus in Toxoplasma gondii Decreases Acute Virulence in Mice Infect Immun. 1998 ,66(9):4176-4182.
[31] Cérède O, Dubremetz JF, So(e|^)te M,etal. Synergistic role of micronemal proteins in Toxoplasma gondii virulence. J Exp Med. 2005,201(3):453-63. Epub 2005 Jan 31.
[32] Titus RG,Gueiros-Filho-FJ,de-Freitas-LA et al. Development of a safe live Leishmania vaccine line by gene replacement. Proc Natl Acad Sci USA, 1995,92(22): 10267-10271
[33] Robert Ménard. Knockout malaria vaccine? Nature , 2005,433, 113-114.
[34] Mueller, A.-K., Labaied, M., Kappe, S. H. I. & Matuschewski, K. Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature,433,164-167 (2005).
[35] Cowman AF,McFadden GI,et al.Dissecting apicoplast targeting in the malaria parasite Plasmodium falciparum. Science,2003,299(5607):705-708.
[36] Engers HD et al. Progress on vaccines against parasites.Dev Biol Stand. 1996,87:73-84. Review.