弓形虫速殖子与缓殖子体外转化体系的建立及BAG1基因在转化中作用的初步研究
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摘要
刚地弓形虫(Toxoplasma gondii)是一种专性细胞内寄生原虫,能感染所有的恒温动物,引起人兽共患的弓形虫病。此虫呈世界性分布,估计人类感染弓形虫的血清学阳性率达到30%。弓形虫是一种机会性致病原虫,在宿主免疫力正常的情况下,大多表现为无临床症状的隐性感染,短则数月,长则数年甚至终生寄生在人体。而当机体免疫功能下降或受抑制(如恶性肿瘤患者、器官移植者及AIDS患者等)时,处于隐性感染状态的虫体就会活化,迅速增生繁殖,破坏宿主细胞,引起多器官的损害,严重者可导致宿主死亡。孕妇感染弓形虫后,除自身患病外,还可通过胎盘将虫体垂直传播给胎儿,从而引起流产、早产、死胎、畸形等,幸存者也常遗留智能低下等严重后遗症。近年来,由于艾滋病的流行和宠物饲养的逐渐增多,弓形虫的危害也日益突出,弓形虫病的防治已成为一个严重的公共卫生问题。
弓形虫感染的特点是机会性致病,其生物学基础是速殖子和缓殖子的相互转换。目前对弓形虫病的治疗仍以药物为主,如乙胺嘧啶、乙酰螺旋霉素、复方新诺明等及一些细胞因子如IFN-γ、IL-2等的单独或联合应用,对速殖子的杀灭、抑制作用较明显,但对包囊内的缓殖子则几乎没有作用。作为弓形虫治疗药物和疫苗研制的重要靶标,弓形虫速殖子与缓殖子相互转化的机制一直是弓形虫研究的热点,建立弓形虫速殖子与缓殖子相互转化的体外模型对于这一机制的研究具有重要意义。经过长期摸索,科学家们发现在细胞培养状况下,通过改变理化(如培养基的温度、pH值或剥夺营养)和生物(如加入IFN-γ或NO)条件,可以实现速殖子与缓殖子的转化,然而,改变外界条件引起的转化过程与自然条件下的转化过程还是存在一定的差距。本实验尝试在尽可能自然的条件下,在COS-7细胞内建立弓形虫速殖子和缓殖子相互转化的体外模型。
弓形虫缓殖子期抗原1(bradyzoite antigen 1, BAG1),是弓形虫缓殖子主要期特异性抗原之一,BAG1在弓形虫速殖子向缓殖子转变的早期被表达,是成熟组织包囊的主要蛋白,但BAG1对此转化过程的影响尚不清楚。近年来,以RNA干扰和转基因技术为基础的反向遗传学的发展,为我们提供了一个从基因变化分析基因功能的方法。本研究将构建好的以弓形虫速殖子期特异抗原SAG1基因启动子驱动的GFP蛋白基因为筛选标志的且靶向BAG1基因的可遗传可诱导的RNAi载体pHANA-hairpin/BAG1导入弓形虫速殖子并筛选出纯的转基因弓形虫品系。本实验构建的RNAi载体内含有BAG1编码基因的颠倒重复序列,在其转录过程中通过分子内杂交,该颠倒重复序列将会折叠形成Hairpin dsRNA。hpRNA最终被Dicer加工成siRNA,通过靶向结合内源性的BAG1 mRNA,从而干扰BAG1基因的功能。本实验RNAi载体中的GFP编码序列在SAG1启动子的驱动下进行表达,主要在速殖子期起作用,在缓殖子期GFP表达减弱或消失。因此,通过绿色荧光的表达强弱可以判断弓形虫的转化状态,并进一步推断BAG1基因对转化过程的影响,以阐明其在速殖子向缓殖子转化过程中的功能和作用。
目的：
1.建立弓形虫速殖子与缓殖子体外转化体系；
2.建立转基因弓形虫品系；
3.探讨弓形虫缓殖子期特异蛋白BAG1基因的功能及在速殖子与缓殖子相互转化中的作用。
方法：
1.复苏液氮冻存的弓形虫RH株速殖子,接种小鼠腹腔,连续传代转种3次后,用生理盐水冲洗小鼠腹腔,纯化收集速殖子。
2.当COS-7细胞密度达到培养瓶的60%-70%时,按照1：10比例接种弓形虫速殖子,置于37℃,5%CO2的培养箱中培养。
3.分别在接种后第1、2、3、4、5、6天观察细胞和虫体的形态并提取每瓶细胞和虫体的Total RNA,用RT-PCR的方法检测速殖子期特异性蛋白SAG1基因、缓殖子期特异性蛋白BAGl基因和SAG2C基因的表达。
4.将构建好的以弓形虫速殖子期特异抗原SAG1基因启动子驱动的GFP蛋白基因为筛选标志的且靶向BAG1基因的RNAi载体pHANA-hairpin/BAG1用电穿孔的方法导入弓形虫RH株速殖子内,扩大培养后,用有限稀释法筛选出纯的转基因弓形虫。
5.将纯化的转基因弓形虫RH株速殖子按已建立好的条件接种于COS-7细胞内进行体外培养。分别在接种后第1、2、3、4、5、6天观察速殖子向缓殖子转变的过程并提取每瓶细胞和虫体的Total RNA,用RT-PCR的方法检测速殖子期特异性蛋白SAG1基因、缓殖子期特异性蛋白BAG1基因和SAG2C基因的表达。
6.将纯化的转基因弓形虫RH株速殖子及普通弓形虫速殖子按已建好的条件接种于COS-7细胞内。在荧光倒置显微镜下分别计算转基因弓形虫及普通弓形虫在1-6天内形成类包囊样结构的数量,收集数据,进行统计分析。
结果：
1.随着速殖子在COS-7细胞中培养时间的延续,虫体的数量逐渐增多,但增殖速度逐步减缓；虫体在细胞内的排列方式也在不断变化,由成对的弧形、玫瑰花形或簇形、变成半圆形最后形成圆形的类包囊样结构。RT-PCR检测显示：速殖子期特异性蛋白SAG1基因在培养的第1-6天均有表达,且表达量呈递增趋势；缓殖子期特异性蛋白BAG1基因从第2天开始出现表达,随时间延续表达量逐渐增加；缓殖子期特异性蛋白SAG2C基因从第5天开始表达,第6天表达量增加。以上结果表明有越来越多的速殖子转化为缓殖子。
2.将RNAi载体用电穿孔的方法导入弓形虫RH株速殖子内,电转24h后即可观察到发绿色荧光的转基因弓形虫。用有限稀释法筛选出纯的转基因弓形虫。
3.利用已建立的弓形虫体外转化模型促使速殖子向缓殖子转化,随着纯化的转基因弓形虫速殖子在COS-7细胞中培养时间的延续,虫体的数量逐渐增多,但增殖速度逐步减缓；虫体在细胞内的排列方式也在不断变化,由成对的弧形、玫瑰花形或簇形、变成半圆形最后形成圆形的类包囊样结构。随着时间的延续,绿色荧光强度略有降低。RT-PCR检测显示：速殖子期特异性蛋白SAG1基因在培养的第1-6天均有表达,且表达量呈递增趋势；缓殖子期特异性蛋白BAG1基因从第5天开始出现表达,随时间延续表达量逐渐增加；缓殖子期特异性蛋白SAG2C基因从第5天开始表达,第6天表达量增加。
4.转基因弓形虫形成的类包囊数与普通弓形虫形成的类包囊数有统计学差异(F=53.263,P<0.001)。数据显示：转基因弓形虫出现类包囊样结构的时间比普通弓形虫晚,且数量也少。
结论：
1.成功构建弓形虫速殖子与缓殖子体外相互转化体系,为其转化机制的研究奠定了基础。
2.成功获得1个转GFP基因的弓形虫品系。
3.干扰弓形虫缓殖子期特异蛋白BAGl基因能使BAG1基因表达时间延迟表达量减少,但不能完全阻止包囊形成。
Background:Toxoplasma gondii is a widespread obligate intracellular protozoan parasite which could infect all species of mammals (including human) and is responsible for toxoplasmosis. It is considered be one of the most ubiquitously distributed and widespread parasites with a worldwide seroprevalence of up to 30% in humans. Toxoplasma gondii is an opportunistic parasite:in an immunocompetent host infected by Toxoplasma gondii, the disease is generally asymptomatic. Parasites can escape immune attack and form cysts which are able to persist in the body of a host for a few months, several years or even a life time. However, as far as immunocompromised or immunodeficiency individuals are concerned, such as patients suffering from neoplastic disease, organ transplantation and AIDS patients et al, T. gondii tachyzoites could multiply rapidly and produces necrotic lesions that may result in serious damage such as toxoplasmic encephalitis, toxoplasmic retinochoroiditis, even death of the patients. Toxoplasma gondii could be transmitted vertically to foetus through placenta and cause premature birth, abortion, fetal death, abnormity or a baby with developmental malformation. During recent years, owing to the increasing number of AIDS patients and pet-keepers, the infection of T. gondii becomes a severe problem to public health.
The interconversion between tachyzoite and bradyzoite is the key to pathogenesis of toxoplasmosis. The mechanism about the interconversion is still unknown. There are not specific medicine for toxoplasmosis. Drugs, such as pyrimethamine, acetyl spiramycin,et al, are widely used to treat toxoplasmosis, but the drugs can only kill or depress tachyzoite without affecting bradyzoites in the tissue cyst. Reactivation of bradyzoite could result in serious damage of patients. The study on the mechanism of the interconversion has become a hot spot, which is important for the development of drugs and vaccines. It is also very significant to develop an in vitro interconversion system for tachyzoite-bradyzoite of Toxoplasma gondii. After a long-term exploration, scientists have found that changing the cell culture conditions--the physical and chemical (such as medium temperature, pH, or denial of nutrients) and biological (eg, by adding IFN-γor NO) conditions can increase the efficacy of bradyzoite development in vitro. However, there is still a certain gap about the transformation between changing the external conditions and maintaining natural conditions. The transformation study is more significant under natural conditions, so the conditions are largely maintained to set up the interconversing system from tachzoites to bradyzoites of T.gondii in COS-7 cells.
Bradyzoite antigen (BAG) 1 is a bradyzoite-specific protein and it is expressed in the earlier period of the interconversion from tachzoites to bradyzoites of T.gondii.and is a main protein of the mature cysts, but the function of the BAG1 gene remains unclear in the interconversion. Recently, with the development in reverse genetics based on RNAi and transgenic techniques, it provides a new way to analyze gene function from gene variation. In this thesis, the vector pHANA-hairpin/BAG1 containing the GFP gene driven by the SAG1 promoter of T. gondii and targeted BAG1 gene is constructed and electroporated into tachyzoites of T. gondii RH strain. Finally a transgenic strain of Toxoplasma gondii is obtained successfully. The construction of the RNAi vector contains inverted repeat sequences of the BAG1 gene. During the transcription process, the inverted repeat sequences will get folded and form Hairpin dsRNA by intramolecular hybridization. HpRNAs eventually form siRNA under the influence of the Dicer enzyme. The siRNA will combine the endogenous BAG1 mRNA and interfere the function of BAG1 gene. Recombinant T. gondii expressing green fluorescent protein (GFP) under control of the SAG1 promoter are generated, which works at the tachyzoite stage, but decreases or disappeare at the bradyzoite stage. Thus, the conversion state of Toxoplasma gondii in terms of the expression of green fluorescence is determined and the function of the BAG1 gene in the process of the differentiation from tachyzoite to bradyzoite is further illuminated.
Objective:
1. To develop the in vitro interconversing system from tachzoites to bradyzoites of Toxoplasma gondii.
2. To construct the transgenic strain of Toxoplasma gondii.
3. Targeted knock down the endogenous bradyzoite-specific gene, BAG1, and analyze its effect on cyst formation.
Methods:
1. Tachyzoites of T. gondii RH strain were inoculated into the peritoneal cavity of mouse, the peritoneal fluid was collected at the 3-4 day after the inoculation.
2. According to the ratio of 1:10, the purified tachyzoites were inoculated into COS-7 cell layer at the point which the grown COS-7 cell layer occupied 60%-70% of the culture surface, using DMEM supplemented with 5% new-born calf serum, the cells were cultured at 37℃, in 5% CO2.
3. The shape of the cultured cells and parasites were observed and their total RNAs were extracted on 1 to 6 days post inoculation respectively. The expression of tachyzoite-specific protein (SAG1) and bradyzoite-specific proteins (BAG1 and SAG2C) were identified by RT-PCR.
4. The vector pHANA-hairpin/BAG1 containing the GFP gene driven by the SAG1 promoter of T. gondii and targeted BAG1 gene was electroporated into tachyzoites of T.gondii RH strain. Individual positive clones were screened by limited dilution.
5. The COS-7 cells were inoculated with the purified transgenic tachyzoites of T.gondii RH strain and cultured under certain condition.The stage conversion from tachzoites to bradyzoites of T.gondii was observed and their total RNAs were extracted on 1 to 6 days post inoculation respectively. The expression of tachyzoite-specific protein (SAG1) and bradyzoite-specific proteins (BAG1 and SAG2C) were identified by RT-PCR. Optimize expression conditions and purify expression products.
6. The COS-7 cells were inoculated with the purified transgenic tachyzoites and tachyzoites of T.gondii RH strain and cultured under certain condition. The encapsulation-like structures were calculated from transgenic parasites and parasites in COS-7 cell on 1 to 6 days through a fluorescence microscope. Then data collection and statistical analysis are carried out.
Results:
1. As time went by, the number of parasites in COS-7 cell increased gradually but the proliferation rate decreased. Intracellular tachyzoites kept breeding in budding mode and binary fission, which led to the changes on arrangement of the parasites in the cells from bigeminal curved, rose-shaped, or cluster-shaped, semi-circular shape to round encapsulation-like structure. These series of changes indicated that the tachyzoites could gradually be transformed into bradyzoites. The expressions of the tachyzoite-specific SAG1 gene could be detected from the first day to the 6th day. The expression of the bradyzoite-specific BAG1 gene was detected since the second day of post inoculation and SAG2C gene was detected since the fifth day. Changing the culture condition, the bradyzoites were able to be gradually transformed into tachyzoites as well.
2. Within 24 hours after the electroporation, the transfected tachyzoites of T.gondii expressing green fluorescence were observed with a fluorescence microscope.
3. As time went by, the number of the purified transgenic tachyzoites in COS-7 cell increased gradually but the proliferation rate decreased. Intracellular tachyzoites kept breeding in budding mode and binary fission, which caused the changes on arrangement of the parasites in the cells from bigeminal curved, rose-shaped, or cluster-shaped, semi-circular shape to round encapsulation-like structure. This series of changes indicated that the tachyzoites could gradually transform into bradyzoites. With time going by, Fluorescence intensity reduced gradually. The expressions of the tachyzoite-specific SAG1 gene could be detected from the first day to the 6th day. The expression of the bradyzoite-specific BAG1 gene had been detected since the fifth day of post inoculation and SAG2C gene had been detected since the fifth day. Targeted disruption of the bradyzoite-specific gene BAG1 could delay the expression of the bradyzoite-specific BAG1 gene rather than stop tissue cyst formation in Toxoplasma gondii.
4、The number of encapsulation-like structure from the transgenic parasites and parasites has statistical significance. (F=53.263, P<0.001). The data states that the encapsulation-like structure from the transgenic parasites appear later and remain less in number compared with ordinary Toxoplasma gondii.
Conclusion:
1. An in vitro interconversing system from tachzoites to bradyzoites of T. gondii is developed successfully in present work, which sets up a good basis for further study on the mechanism of interconversion.
2. A transgenic strain of Toxoplasma gondii is obtained successfully.
3. Targeted disruption of the bradyzoite-specific gene BAG1 could not stop tissue cyst formation in Toxoplasma gondii.
弓形虫感染的特点是机会性致病,其生物学基础是速殖子和缓殖子的相互转换。目前对弓形虫病的治疗仍以药物为主,如乙胺嘧啶、乙酰螺旋霉素、复方新诺明等及一些细胞因子如IFN-γ、IL-2等的单独或联合应用,对速殖子的杀灭、抑制作用较明显,但对包囊内的缓殖子则几乎没有作用。作为弓形虫治疗药物和疫苗研制的重要靶标,弓形虫速殖子与缓殖子相互转化的机制一直是弓形虫研究的热点,建立弓形虫速殖子与缓殖子相互转化的体外模型对于这一机制的研究具有重要意义。经过长期摸索,科学家们发现在细胞培养状况下,通过改变理化(如培养基的温度、pH值或剥夺营养)和生物(如加入IFN-γ或NO)条件,可以实现速殖子与缓殖子的转化,然而,改变外界条件引起的转化过程与自然条件下的转化过程还是存在一定的差距。本实验尝试在尽可能自然的条件下,在COS-7细胞内建立弓形虫速殖子和缓殖子相互转化的体外模型。
弓形虫缓殖子期抗原1(bradyzoite antigen 1, BAG1),是弓形虫缓殖子主要期特异性抗原之一,BAG1在弓形虫速殖子向缓殖子转变的早期被表达,是成熟组织包囊的主要蛋白,但BAG1对此转化过程的影响尚不清楚。近年来,以RNA干扰和转基因技术为基础的反向遗传学的发展,为我们提供了一个从基因变化分析基因功能的方法。本研究将构建好的以弓形虫速殖子期特异抗原SAG1基因启动子驱动的GFP蛋白基因为筛选标志的且靶向BAG1基因的可遗传可诱导的RNAi载体pHANA-hairpin/BAG1导入弓形虫速殖子并筛选出纯的转基因弓形虫品系。本实验构建的RNAi载体内含有BAG1编码基因的颠倒重复序列,在其转录过程中通过分子内杂交,该颠倒重复序列将会折叠形成Hairpin dsRNA。hpRNA最终被Dicer加工成siRNA,通过靶向结合内源性的BAG1 mRNA,从而干扰BAG1基因的功能。本实验RNAi载体中的GFP编码序列在SAG1启动子的驱动下进行表达,主要在速殖子期起作用,在缓殖子期GFP表达减弱或消失。因此,通过绿色荧光的表达强弱可以判断弓形虫的转化状态,并进一步推断BAG1基因对转化过程的影响,以阐明其在速殖子向缓殖子转化过程中的功能和作用。
目的：
1.建立弓形虫速殖子与缓殖子体外转化体系；
2.建立转基因弓形虫品系；
3.探讨弓形虫缓殖子期特异蛋白BAG1基因的功能及在速殖子与缓殖子相互转化中的作用。
方法：
1.复苏液氮冻存的弓形虫RH株速殖子,接种小鼠腹腔,连续传代转种3次后,用生理盐水冲洗小鼠腹腔,纯化收集速殖子。
2.当COS-7细胞密度达到培养瓶的60%-70%时,按照1：10比例接种弓形虫速殖子,置于37℃,5%CO2的培养箱中培养。
3.分别在接种后第1、2、3、4、5、6天观察细胞和虫体的形态并提取每瓶细胞和虫体的Total RNA,用RT-PCR的方法检测速殖子期特异性蛋白SAG1基因、缓殖子期特异性蛋白BAGl基因和SAG2C基因的表达。
4.将构建好的以弓形虫速殖子期特异抗原SAG1基因启动子驱动的GFP蛋白基因为筛选标志的且靶向BAG1基因的RNAi载体pHANA-hairpin/BAG1用电穿孔的方法导入弓形虫RH株速殖子内,扩大培养后,用有限稀释法筛选出纯的转基因弓形虫。
5.将纯化的转基因弓形虫RH株速殖子按已建立好的条件接种于COS-7细胞内进行体外培养。分别在接种后第1、2、3、4、5、6天观察速殖子向缓殖子转变的过程并提取每瓶细胞和虫体的Total RNA,用RT-PCR的方法检测速殖子期特异性蛋白SAG1基因、缓殖子期特异性蛋白BAG1基因和SAG2C基因的表达。
6.将纯化的转基因弓形虫RH株速殖子及普通弓形虫速殖子按已建好的条件接种于COS-7细胞内。在荧光倒置显微镜下分别计算转基因弓形虫及普通弓形虫在1-6天内形成类包囊样结构的数量,收集数据,进行统计分析。
结果：
1.随着速殖子在COS-7细胞中培养时间的延续,虫体的数量逐渐增多,但增殖速度逐步减缓；虫体在细胞内的排列方式也在不断变化,由成对的弧形、玫瑰花形或簇形、变成半圆形最后形成圆形的类包囊样结构。RT-PCR检测显示：速殖子期特异性蛋白SAG1基因在培养的第1-6天均有表达,且表达量呈递增趋势；缓殖子期特异性蛋白BAG1基因从第2天开始出现表达,随时间延续表达量逐渐增加；缓殖子期特异性蛋白SAG2C基因从第5天开始表达,第6天表达量增加。以上结果表明有越来越多的速殖子转化为缓殖子。
2.将RNAi载体用电穿孔的方法导入弓形虫RH株速殖子内,电转24h后即可观察到发绿色荧光的转基因弓形虫。用有限稀释法筛选出纯的转基因弓形虫。
3.利用已建立的弓形虫体外转化模型促使速殖子向缓殖子转化,随着纯化的转基因弓形虫速殖子在COS-7细胞中培养时间的延续,虫体的数量逐渐增多,但增殖速度逐步减缓；虫体在细胞内的排列方式也在不断变化,由成对的弧形、玫瑰花形或簇形、变成半圆形最后形成圆形的类包囊样结构。随着时间的延续,绿色荧光强度略有降低。RT-PCR检测显示：速殖子期特异性蛋白SAG1基因在培养的第1-6天均有表达,且表达量呈递增趋势；缓殖子期特异性蛋白BAG1基因从第5天开始出现表达,随时间延续表达量逐渐增加；缓殖子期特异性蛋白SAG2C基因从第5天开始表达,第6天表达量增加。
4.转基因弓形虫形成的类包囊数与普通弓形虫形成的类包囊数有统计学差异(F=53.263,P<0.001)。数据显示：转基因弓形虫出现类包囊样结构的时间比普通弓形虫晚,且数量也少。
结论：
1.成功构建弓形虫速殖子与缓殖子体外相互转化体系,为其转化机制的研究奠定了基础。
2.成功获得1个转GFP基因的弓形虫品系。
3.干扰弓形虫缓殖子期特异蛋白BAGl基因能使BAG1基因表达时间延迟表达量减少,但不能完全阻止包囊形成。
Background:Toxoplasma gondii is a widespread obligate intracellular protozoan parasite which could infect all species of mammals (including human) and is responsible for toxoplasmosis. It is considered be one of the most ubiquitously distributed and widespread parasites with a worldwide seroprevalence of up to 30% in humans. Toxoplasma gondii is an opportunistic parasite:in an immunocompetent host infected by Toxoplasma gondii, the disease is generally asymptomatic. Parasites can escape immune attack and form cysts which are able to persist in the body of a host for a few months, several years or even a life time. However, as far as immunocompromised or immunodeficiency individuals are concerned, such as patients suffering from neoplastic disease, organ transplantation and AIDS patients et al, T. gondii tachyzoites could multiply rapidly and produces necrotic lesions that may result in serious damage such as toxoplasmic encephalitis, toxoplasmic retinochoroiditis, even death of the patients. Toxoplasma gondii could be transmitted vertically to foetus through placenta and cause premature birth, abortion, fetal death, abnormity or a baby with developmental malformation. During recent years, owing to the increasing number of AIDS patients and pet-keepers, the infection of T. gondii becomes a severe problem to public health.
The interconversion between tachyzoite and bradyzoite is the key to pathogenesis of toxoplasmosis. The mechanism about the interconversion is still unknown. There are not specific medicine for toxoplasmosis. Drugs, such as pyrimethamine, acetyl spiramycin,et al, are widely used to treat toxoplasmosis, but the drugs can only kill or depress tachyzoite without affecting bradyzoites in the tissue cyst. Reactivation of bradyzoite could result in serious damage of patients. The study on the mechanism of the interconversion has become a hot spot, which is important for the development of drugs and vaccines. It is also very significant to develop an in vitro interconversion system for tachyzoite-bradyzoite of Toxoplasma gondii. After a long-term exploration, scientists have found that changing the cell culture conditions--the physical and chemical (such as medium temperature, pH, or denial of nutrients) and biological (eg, by adding IFN-γor NO) conditions can increase the efficacy of bradyzoite development in vitro. However, there is still a certain gap about the transformation between changing the external conditions and maintaining natural conditions. The transformation study is more significant under natural conditions, so the conditions are largely maintained to set up the interconversing system from tachzoites to bradyzoites of T.gondii in COS-7 cells.
Bradyzoite antigen (BAG) 1 is a bradyzoite-specific protein and it is expressed in the earlier period of the interconversion from tachzoites to bradyzoites of T.gondii.and is a main protein of the mature cysts, but the function of the BAG1 gene remains unclear in the interconversion. Recently, with the development in reverse genetics based on RNAi and transgenic techniques, it provides a new way to analyze gene function from gene variation. In this thesis, the vector pHANA-hairpin/BAG1 containing the GFP gene driven by the SAG1 promoter of T. gondii and targeted BAG1 gene is constructed and electroporated into tachyzoites of T. gondii RH strain. Finally a transgenic strain of Toxoplasma gondii is obtained successfully. The construction of the RNAi vector contains inverted repeat sequences of the BAG1 gene. During the transcription process, the inverted repeat sequences will get folded and form Hairpin dsRNA by intramolecular hybridization. HpRNAs eventually form siRNA under the influence of the Dicer enzyme. The siRNA will combine the endogenous BAG1 mRNA and interfere the function of BAG1 gene. Recombinant T. gondii expressing green fluorescent protein (GFP) under control of the SAG1 promoter are generated, which works at the tachyzoite stage, but decreases or disappeare at the bradyzoite stage. Thus, the conversion state of Toxoplasma gondii in terms of the expression of green fluorescence is determined and the function of the BAG1 gene in the process of the differentiation from tachyzoite to bradyzoite is further illuminated.
Objective:
1. To develop the in vitro interconversing system from tachzoites to bradyzoites of Toxoplasma gondii.
2. To construct the transgenic strain of Toxoplasma gondii.
3. Targeted knock down the endogenous bradyzoite-specific gene, BAG1, and analyze its effect on cyst formation.
Methods:
1. Tachyzoites of T. gondii RH strain were inoculated into the peritoneal cavity of mouse, the peritoneal fluid was collected at the 3-4 day after the inoculation.
2. According to the ratio of 1:10, the purified tachyzoites were inoculated into COS-7 cell layer at the point which the grown COS-7 cell layer occupied 60%-70% of the culture surface, using DMEM supplemented with 5% new-born calf serum, the cells were cultured at 37℃, in 5% CO2.
3. The shape of the cultured cells and parasites were observed and their total RNAs were extracted on 1 to 6 days post inoculation respectively. The expression of tachyzoite-specific protein (SAG1) and bradyzoite-specific proteins (BAG1 and SAG2C) were identified by RT-PCR.
4. The vector pHANA-hairpin/BAG1 containing the GFP gene driven by the SAG1 promoter of T. gondii and targeted BAG1 gene was electroporated into tachyzoites of T.gondii RH strain. Individual positive clones were screened by limited dilution.
5. The COS-7 cells were inoculated with the purified transgenic tachyzoites of T.gondii RH strain and cultured under certain condition.The stage conversion from tachzoites to bradyzoites of T.gondii was observed and their total RNAs were extracted on 1 to 6 days post inoculation respectively. The expression of tachyzoite-specific protein (SAG1) and bradyzoite-specific proteins (BAG1 and SAG2C) were identified by RT-PCR. Optimize expression conditions and purify expression products.
6. The COS-7 cells were inoculated with the purified transgenic tachyzoites and tachyzoites of T.gondii RH strain and cultured under certain condition. The encapsulation-like structures were calculated from transgenic parasites and parasites in COS-7 cell on 1 to 6 days through a fluorescence microscope. Then data collection and statistical analysis are carried out.
Results:
1. As time went by, the number of parasites in COS-7 cell increased gradually but the proliferation rate decreased. Intracellular tachyzoites kept breeding in budding mode and binary fission, which led to the changes on arrangement of the parasites in the cells from bigeminal curved, rose-shaped, or cluster-shaped, semi-circular shape to round encapsulation-like structure. These series of changes indicated that the tachyzoites could gradually be transformed into bradyzoites. The expressions of the tachyzoite-specific SAG1 gene could be detected from the first day to the 6th day. The expression of the bradyzoite-specific BAG1 gene was detected since the second day of post inoculation and SAG2C gene was detected since the fifth day. Changing the culture condition, the bradyzoites were able to be gradually transformed into tachyzoites as well.
2. Within 24 hours after the electroporation, the transfected tachyzoites of T.gondii expressing green fluorescence were observed with a fluorescence microscope.
3. As time went by, the number of the purified transgenic tachyzoites in COS-7 cell increased gradually but the proliferation rate decreased. Intracellular tachyzoites kept breeding in budding mode and binary fission, which caused the changes on arrangement of the parasites in the cells from bigeminal curved, rose-shaped, or cluster-shaped, semi-circular shape to round encapsulation-like structure. This series of changes indicated that the tachyzoites could gradually transform into bradyzoites. With time going by, Fluorescence intensity reduced gradually. The expressions of the tachyzoite-specific SAG1 gene could be detected from the first day to the 6th day. The expression of the bradyzoite-specific BAG1 gene had been detected since the fifth day of post inoculation and SAG2C gene had been detected since the fifth day. Targeted disruption of the bradyzoite-specific gene BAG1 could delay the expression of the bradyzoite-specific BAG1 gene rather than stop tissue cyst formation in Toxoplasma gondii.
4、The number of encapsulation-like structure from the transgenic parasites and parasites has statistical significance. (F=53.263, P<0.001). The data states that the encapsulation-like structure from the transgenic parasites appear later and remain less in number compared with ordinary Toxoplasma gondii.
Conclusion:
1. An in vitro interconversing system from tachzoites to bradyzoites of T. gondii is developed successfully in present work, which sets up a good basis for further study on the mechanism of interconversion.
2. A transgenic strain of Toxoplasma gondii is obtained successfully.
3. Targeted disruption of the bradyzoite-specific gene BAG1 could not stop tissue cyst formation in Toxoplasma gondii.
引文
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