【作者】 谭逵；

【导师】 舒衡平；

【作者基本信息】 中南大学 ， 微生物学， 2008， 硕士

【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。更多还原

【Abstract】 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.更多还原