弓形虫速殖子与缓殖子体外相互转化模型的建立及可调性RNA干扰的初步研究
发布时间：2018-07-25 14:22
【摘要】：刚地弓形虫(Toxoplasma gondii)是一种呈世界性分布的专性细胞内寄生原虫，可寄生在除红细胞外的几乎所有有核细胞中，引起人兽共患的弓形虫病。弓形虫是一种机会性致病原虫，机体免疫功能正常者感染弓形虫一般没有明显症状，为隐性感染状态。但对于免疫功能受损或缺陷者，如肿瘤病人，AIDS患者等，弓形虫在患者体内可以大量增殖，引起全身播散性损害，严重者可导致死亡。此外，妊娠期感染弓形虫可通过胎盘垂直传播，引起早产、流产、死胎、畸胎或婴儿发育畸形等。近年来，，由于艾滋病的广泛流行和宠物饲养的逐渐增多，弓形虫的危害也日益突出，弓形虫感染成为一个严重的公共卫生问题。 弓形虫速殖子与缓殖子的相互转化，是弓形虫致病的中心环节。速殖子可引起宿主的急性感染，而包囊内的缓殖子是形成慢性感染的主要形式，弓形虫速殖子与缓殖子相互转化的机制尚不清楚。目前弓形虫病的治疗尚无特效药物，主要以乙胺嘧啶，乙酰螺旋霉素等药物为主，这些药物对速殖子有杀灭、抑制作用，而对包囊内的缓殖子几乎没有作用。但包囊内缓殖子的活化，能对宿主造成极大的危害。因此，探讨弓形虫速殖子与缓殖子相互转换机制的研究成为弓形虫病研究的热点和难点之一。 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。本研究综合目前弓形虫速殖子与缓殖子相互转化的研究现状及研究中存在的主要问题，采用Transgenesis、RNAi和Reverse genetics的原理和方法，建立弓形虫RH株速殖子与缓殖子体外相互转化的细胞模型，克隆转化早期差显基因的编码序列，构建弓形虫热休克蛋白70(HSP70)基因启动子驱动的反向重复序列RNAi载体系统，建立一套进行弓形虫基因功能分析的Reverse Genetics技术平台，并选取在速殖子向缓殖子转化早期开始表达的基因进行重点分析，以阐明这些分子在速殖子向缓殖子转化过程中的功能与作用。 一、弓形虫体外速殖子与缓殖子相互转化模型的建立 小鼠腹腔接种弓形虫RH株速殖子，3-4天后收集小鼠腹腔液，用国产4.5号针头注射器反复抽吸虫体悬液破裂宿主细胞释放虫体，虫体悬液用孔径3μm的滤膜过滤纯化速殖子，速殖子纯度可达98％以上。 将纯化的弓形虫RH株速殖子按照1：10的比例，接种于NIH3T3细胞单层(NIH3T3细胞单层生长达到60％-70％时接种速殖子，培养基含5％新生牛血清)，置于37℃，5％CO_2的培养箱中培养，速殖子增殖迅速，大量破坏宿主细胞，培养5-6天后收集弓形虫速殖子，进行分瓶培养或实验研究。 为探索碱性条件下弓形虫速殖子向缓殖子的诱导转化，按照1：10比例将RH株速殖子接种NIH3T3细胞单层，接种4h后，用pH8.1的DMEM培养基轻轻冲洗细胞单层以移去未侵入细胞的弓形虫速殖子，再加入pH8.1的DMEM培养基(含2.5％新生牛血清)置于37℃空气中培养96h。镜下观察可见NIH3T3细胞胞浆中出现大小不等的圆形囊性小体，囊壁折光性较强。收集培养虫体提取总RNA，采用RT-PCR的方法进行缓殖子期特异抗原缓殖子抗原1(BAG1)mRNA的检测，结果得到700bp左右目的条带，PCR产物克隆到T载体后测序，测序结果与已发表的BAG1基因mRNA同源性高达99.3％，无基因组DNA内含子序列，结果表明成功诱导缓殖子的形成。为监测速殖子向缓殖子转化进程，在诱导第0、24、48、72、96小时，分别取出一瓶培养虫体，提取总RNA，以弓形虫β—微管蛋白的mRNA RT—PCR产物为参照，进行半定量RT—PCR扩增BAG1基因mRNA，结果显示，随着诱导时间的延长，BAG1 mRNA的转录水平逐渐提高，表明随着诱导时间的延伸，有更多的弓形虫速殖子转化成缓殖子。为诱导缓殖子向速殖子的转化，将pH8.1碱性条件诱导形成的缓殖子恢复pH7.2培养基及正常培养条件，24h后可见溶液中出现游离弓形虫速殖子，2-3天后速殖子开始大量增殖，需适当补充NIH3T3细胞才能继续培养。 为探索热休克对弓形虫速殖子向缓殖子的转化影响，将常规培养的NIH3T3细胞分别放入37℃，39℃，41℃，43℃，5％CO_2培养箱中培养3h后，按照1：10比例接种纯化的RH株速殖子，接种4h后，用相应温度预热的DMEM培养基轻轻冲洗细胞单层，以移去未侵入细胞的弓形虫速殖子后，分别放置在37℃，39℃，41℃，43℃，5％CO_2培养箱中培养48h，收集培养混合物，提取总RNA，进行RT-RCR。结果表明39℃诱导条件下，速殖子增殖较快，不能诱导形成缓殖子，39℃诱导条件还是适于速殖子的生长条件。43℃诱导条件下，NIH3T3宿主细胞难以生长，不能为弓形虫速殖子提供一个稳定的细胞内环境，不利于弓形虫RH株速殖子向缓殖子的转化。41℃诱导条件下，NIH3T3宿主细胞变性坏死相对较慢，能为弓形虫速殖子提供一个相对稳定的细胞内环境，弓形虫RH株速殖子能诱导转化为缓殖子。 二、弓形虫可遗传及可诱导的RNAi载体系统的构建 1．弓形虫主要表面抗原1(SAG1)基因启动子驱动的绿色荧光蛋白基因载体的构建：构建该载体的目的是一方面作为转基因弓形虫的一种荧光筛选标志，另一方面作为转基因弓形虫是处于速殖子或缓殖子，还是两者之间中间状态的一种指示。设计引物，通过PCR分别扩增SAG1基因5'UTR启动子序列(SAG1／5UTR)、SAG1基因3'UTR序列(SAG1／3UTR)及绿色荧光蛋白编码序列(eGFP)，通过酶切连接，构建弓形虫SAG1基因启动子驱动的绿色荧光蛋白基因载体pBSK-SAG1／5UTR-eGFP-SAG1／3UTR(pBSK-SAG1／GFP)，序列测定结果正确。 2．弓形虫可诱导的反向重复序列RNAi载体的构建：设计引物，通过PCR分别扩增弓形虫HSP70基因5'UTR启动子序列(HSP70／5UTR)、HSP70基因3'UTR序列(HSP70／3UTR)及β-微管蛋白基因内含子C序列(IntronC)，通过酶切连接，构建以弓形虫热休克蛋白HSP70基因启动子进行驱动的，以β-微管蛋白基因内含子C序列作为间隔序列，以HSP70基因3'UTR序列作为转录终止信号的反向重复序列RNAi载体pBSK-HSP70／5UTR-IntronC-HSP70／3UTR，序列测定结果正确。 3．弓形虫可遗传及可诱导的RNAi载体系统的构建：利用载体pHANA-0.5具有弓形虫穿梭载体的功能，将载体pBSK-SAG1／GFP中的SAG1／5UTR-eGFP-SAG1／3UTR片段克隆到载体pBSK-HSP70／5UTR-IntronC-HSP70／3UTR中形成载体pBSK-GFP-Hairpin，再将该载体中的GFP-Hairpin片段克隆到载体pHANA-0.5中形成弓形虫可遗传及可诱导的RNAi载体系统pHANA-hairpin，序列测定结果正确。 4．弓形虫SAG1基因RNAi载体的构建：为对弓形虫SAG1基因进行RNAi，设计引物，通过PCR分别扩增SAG1基因的正向和反向序列，通过酶切连接，将正向和反向序列克隆到载体pHANA-hairpin相应酶切位点，构建SAG1基因RNAi载体pHANA-hairpin／SAG1，序列测定结果正确。 5．弓形虫BAGl基因RNAi载体的构建：为对弓形虫BAG1基因进行RNAi，设计引物，通过PCR分别扩增BAG1基因的正向和反向序列，通过酶切连接，将正向和反向序列克隆到载体pHANA-hairpin相应酶切位点，构建BAG1基因RNAi载体pHANA-hairpin／BAG1，序列测定结果正确。 三、转基因弓形虫品系的建立及RNAi初步研究 分别将质粒pHANA-hairpin／SAG1和pHANA-hairpin／BAG1通过电转化导入弓形虫RH株速殖子获得两个转基因弓形虫品系。将1X10~7弓形虫速殖子和质粒DNA 10μg重悬于800μl电击缓冲液cytomix中。在4mm间隙电穿孔杯中以电压0.4kv，电容800μF电击1次，时间延迟为6-9msec。转染弓形虫培养24h后，在荧光倒置显微镜下观察，两个品系转基因弓形虫均可观察到绿色荧光的表达，分别提取两个品系转基因弓形虫速殖子总RNA，进行RT-PCR，结果均能检测到GFP基因的转录，证明质粒成功导入弓形虫速殖子并进行了表达。将两个品系转基因弓形虫速殖子进行碱性环境(pH8．1)诱导，诱导培养96h后，在荧光倒置显微镜下观察，均可观测到有绿色荧光表达，分别提取两个品系转基因弓形虫诱导后虫体总RNA，进行RT-PCR，结果均能检测到SAG1、BAG1及GFP基因的转录。出现这种现象可能的原因有：1．部分转基因弓形虫速殖子未转化为缓殖子；2．部分转化的转基因弓形虫处于一种速殖子与缓殖子的中间状态，未形成成熟的缓殖子和包囊；3．诱导干扰的SAG1基因或BAG1基因可能对速殖子向缓殖子转化过程有影响，使得转基因弓形虫仍处于速殖子状态，这些问题尚需进一步研究。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a cosmopolitan parasitic protozoa that can parasitism in almost all nucleated cells except red blood cells, causing zoonosis in human zoonosis. Toxoplasma gondii is a opportunistic pathogenic protozoa, and the immune function of the body is often infected by Toxoplasma gondii. But for persons with impaired or defective immune function, such as cancer patients and AIDS patients, Toxoplasma gondii can proliferate in a large number of patients, cause systemic disseminated damage and cause death. In addition, Toxoplasma gondii can be transmitted vertically through the placenta, causing premature birth, abortion, stillbirth, Teratosis or infant developmental malformation. In recent years, due to the widespread epidemic of AIDS and the increase of pet raising, the harm of Toxoplasma gondii has become increasingly prominent, and Toxoplasma infection has become a serious public health problem.
The mutual transformation between the tachyonus and the retarded Toxoplasma gondii is the central link of the Toxoplasma gondii. The tachyonus can cause acute infection of the host, and the slow colonies in the capsule are the main forms of the chronic infection. The mechanism of the mutual transformation between the tachyonus and the gonogoniosis is unclear. There is no special drug for the treatment of toxoplasmosis before the eye. Drugs such as ethamimidine and acetyl spiramycin are the main drugs, these drugs kill and inhibit the tachyonus, but have little effect on the retardant in the capsule. But the activation of the retarded colonies in the capsule can cause great harm to the host. Therefore, the study of the mutual conversion mechanism between the tachyonus and the retardant of the Toxoplasma gondii has become the research of the toxoplasmosis. One of the hotspots and difficulties in the study.
In recent years, the development of reverse genetics based on RNA interference and transgenic technology has provided us with a method to analyze gene function from gene change. This study synthesizes the current research status of the mutual transformation of Toxoplasma gondii and retarded colonies and the main problems in the study, using Transgenesis, RNAi and Reverse genetics. The principle and method of the cell model of the mutual transformation between the tachyonus RH of Toxoplasma gondii and the retarded colonies in vitro, clone the coding sequence of the early differential gene and construct the reverse repeat sequence RNAi vector driven by the heat shock protein 70 (HSP70) gene promoter of Toxoplasma gondii, and build a set of Reverse Genet for the analysis of the gene function of Toxoplasma gondii. The ICs technology platform was used to analyze the genes that the tachyonus began to express early in the transformation of the retarded colonies to clarify the function and function of these molecules in the transformation of the tachyonite to the retarded colonies.
1. Establishment of an in vitro transformation model between tachyzoites and bradyzoites of Toxoplasma gondii
The mice intraperitoneally inoculated the tachyonus RH of Toxoplasma gondii, after 3-4 days, the mouse peritoneal fluid was collected. The host cells were released from the host cells of the domestic 4.5 needle syringes repeatedly sucking the sucking body suspension. The parasite suspension was filtered by the filter membrane with the diameter of 3 micron m. The purity of the tachyonus could reach more than 98%.
The purified Toxoplasma gondii RH strain tachyonus was inoculated in the NIH3T3 cell monolayer (NIH3T3 cell monolayer growth reached 60%-70%, inoculated tachyonus, culture medium containing 5% newborn bovine serum), cultured in 37 centigrade, 5%CO_2 culture box, rapid proliferation of tachyonite, massive destruction of host cells, and culturing Toxoplasma gondii in 5-6 days. A tachyonus, flask culture or experimental study.
In order to explore the induction and transformation of tachyonus from Toxoplasma gondii to retarded colonies under alkaline conditions, RH strain tachyonus was inoculated NIH3T3 cell monolayer according to the proportion of 1:10. After inoculation of 4h, the cell monolayer was gently flushed with pH8.1 DMEM medium to remove the Toxoplasma gondii tachyonus from the non invasive cells, and then the DMEM medium of pH8.1 (containing 2.5% newborn bovine serum) was placed in 3. The circular cystic corpuscles in the cytoplasm of NIH3T3 cells were observed under the 96h. microscope at 7 C, and the cysts of different sizes were found in the cytoplasm of the cells. The cyst wall was highly refracted. The total RNA was extracted and cultured. The method of RT-PCR was used to detect the retarded antigen 1 (BAG1) mRNA of the slow colonization antigen, and the 700bp target bands were obtained, and the PCR products were cloned to T. After the sequencing, the homology of the published BAG1 gene mRNA was 99.3%, and the sequence of the DNA intron was no genome. The results showed that the formation of the retarded colonies was successfully induced. In order to monitor the transformation process of the tachyonus to the retarded colonies, a bottle of the cultured insect body was extracted, the total RNA was extracted, and the Toxoplasma beta microtubule eggs were extracted from the induction of the 0,24,48,72,96 hour. The mRNA RT - PCR product of white was used as a reference to amplify the BAG1 gene mRNA by semi quantitative RT PCR. The result showed that the transcription level of BAG1 mRNA increased gradually with the prolongation of induction time, indicating that more Toxoplasma tachycolonies were converted to retarded colonies with the extension of induction time. To induce the transformation of the slow colonies to the tachyonite, the pH8.1 alkaline strip was used. The pH7.2 medium and normal culture conditions were restored by the induced colonies. After 24h, the tachyonus of the Toxoplasma gondii was found in the solution. After 2-3 days, the tachyonus began to proliferate. It was necessary to supplement the NIH3T3 cells to continue the culture.
In order to explore the effect of heat shock on the transformation of Toxoplasma gondii to the retarded colonies, the conventional cultured NIH3T3 cells were put into 37, 39, 41, 43, and 5%CO_2 culture incubator for 3h, and the purified RH tachyonus was inoculated in proportion to the 1:10 proportion. After inoculation of 4h, the cell monolayer was gently flushed with the corresponding temperature preheated DMEM medium. After intruding the tachyonus of Toxoplasma gondii, they were placed at 37, 39, 41, 43, respectively, and cultured in 5%CO_2 incubator for 48h. The culture mixture was collected and the total RNA was extracted. The results of RT-RCR. showed that the tachyonite could not be induced to form a retarder under the condition of 39 degrees centigrade. The induction condition of the tachyonite was.43 centigrade for the growth condition of the tachyonite. Under the guidance conditions, NIH3T3 host cells are difficult to grow, and can not provide a stable intracellular environment for Toxoplasma tachygonite, which is not conducive to the transformation of Toxoplasma RH tachyonus to the retarded colonies at.41 C, and the degeneration and necrosis of the host cells of the host cell are relatively slow, and can provide a relatively stable intracellular environment for the Toxoplasma tachyonus, and the Toxoplasma gondii The tachyonus of RH strain can be induced to be retarded.
2. Construction of a Genetically and Inducible RNAi Vector System of Toxoplasma gondii
The construction of a green fluorescent protein gene carrier driven by the main surface antigen 1 (SAG1) gene promoter of Toxoplasma gondii: the purpose of building the vector is one of the fluorescent screening markers of the transgenic Toxoplasma gondii, on the other hand, as a transgenic Toxoplasma gondii is a tachyonite or a slow colonization, or a intermediate state between the two. Primers were designed to amplify the SAG1 gene 5'UTR promoter sequence (SAG1 / 5UTR), SAG1 gene 3'UTR sequence (SAG1 / 3UTR) and green fluorescent protein coding sequence (eGFP) respectively, and the green fluorescent protein gene carrier driven by the promoter of Toxoplasma gondii SAG1 gene promoter was constructed by PCR. P), the result of sequence determination is correct.
2. toxoplasmosis inducible reverse repeat sequence RNAi vector construction: primers were designed to amplify the 5'UTR promoter sequence (HSP70 / 5UTR) of Toxoplasma gondii HSP70 gene (HSP70 / 5UTR), HSP70 gene 3'UTR sequence (HSP70 / 3UTR) and beta microtubulin gene intron C sequence (IntronC), and the Toxoplasma gondii heat shock protein was constructed by enzyme digestion. The gene promoter was driven by using the intron C sequence of the beta tubulin gene as the interval sequence and the HSP70 gene 3'UTR sequence as the reverse repeat sequence RNAi vector pBSK-HSP70 / 5UTR-IntronC-HSP70 / 3UTR for the transcription termination signal, and the sequencing results were correct.
3. Toxoplasma gondii can be inherited and inducible RNAi vector system construction: the carrier pHANA-0.5 has the function of the Toxoplasma gondii shuttle carrier, and the SAG1 / 5UTR-eGFP-SAG1 / 3UTR fragment in the carrier pBSK-SAG1 / GFP is cloned into the carrier pBSK-HSP70 / 5UTR-IntronC-HSP70 / 3UTR to form carrier pBSK-GFP-Hairpin, and then the GFP-Ha in the carrier The irpin fragment was cloned into the vector pHANA-0.5 to form the inheritable and inducible RNAi vector system pHANA-hairpin of Toxoplasma gondii. The sequencing results were correct.
4. SAG1 gene RNAi vector of Toxoplasma gondii: to RNAi the SAG1 gene of Toxoplasma gondii, design primers, amplify the positive and reverse sequence of SAG1 gene by PCR, and clone the positive and reverse sequences to the corresponding enzyme tangent site of the carrier pHANA-hairpin through PCR, and construct the pHANA-hairpin / SAG1, sequence determination of the RNAi carrier of the SAG1 gene. The result is correct.
5. BAGl gene RNAi vector of Toxoplasma gondii: to RNAi the BAG1 gene of Toxoplasma gondii, design primers, amplify the positive and reverse sequence of BAG1 gene by PCR, and clone the positive and reverse sequences to the corresponding enzyme tangent site of the carrier pHANA-hairpin through PCR, and construct the pHANA-hairpin / BAG1, sequence determination of the RNAi carrier of the BAG1 gene. The result is correct.
Establishment of transgenic Toxoplasma gondii strains and preliminary study on RNAi
Two transgenic Toxoplasma gondii strains were obtained by converting plasmids pHANA-hairpin / SAG1 and pHANA-hairpin / BAG1 to Toxoplasma gondii tachygonite by electrical transformation. The tachygonite and plasmid DNA 10 u g were suspended in the 800 Mug shock buffer liquid cytomix. The voltage 0.4kV in the 4mm gap electroporation cup, the capacitance of 800 mu 1 times, and the time delay. After 24h transfection of Toxoplasma gondii transfected with 6-9msec., the expression of green fluorescence was observed in two strains of transgenic Toxoplasma gondii, and two strains of transgenic Toxoplasma gondii tachygonite total RNA were extracted, and RT-PCR was carried out respectively. The results showed that the transcription of the GFP gene could be detected and the plasmid was successfully introduced into the tachygonite. The expression of two strains of transgenic Toxoplasma gondii was induced by alkaline environment (pH8.1). After induction and culture of 96h, green fluorescence was observed under the fluorescence inversion microscope, and two strains of transgenic Toxoplasma gondii induced total RNA and RT-PCR were extracted respectively, and all of them could detect SAG1, BAG1 and GFP base. The possible causes of this phenomenon are as follows: 1. parts of the transgenic Toxoplasma gondii have not been transformed into the retarded colonies; the 2. transformation of the transgenic Toxoplasma gondii is in the middle state of a tachyonite and the retarded colonies, and does not form mature retarded colonies and cysts; 3. induced interference with the SAG1 gene or BAG1 gene may be a tachyonite. Transgenic Toxoplasma gondii is still in tachyzoite state because of the influence of the transformation process to bradyzoites. These problems need further study.
【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R383
本文编号：2144097
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a cosmopolitan parasitic protozoa that can parasitism in almost all nucleated cells except red blood cells, causing zoonosis in human zoonosis. Toxoplasma gondii is a opportunistic pathogenic protozoa, and the immune function of the body is often infected by Toxoplasma gondii. But for persons with impaired or defective immune function, such as cancer patients and AIDS patients, Toxoplasma gondii can proliferate in a large number of patients, cause systemic disseminated damage and cause death. In addition, Toxoplasma gondii can be transmitted vertically through the placenta, causing premature birth, abortion, stillbirth, Teratosis or infant developmental malformation. In recent years, due to the widespread epidemic of AIDS and the increase of pet raising, the harm of Toxoplasma gondii has become increasingly prominent, and Toxoplasma infection has become a serious public health problem.
The mutual transformation between the tachyonus and the retarded Toxoplasma gondii is the central link of the Toxoplasma gondii. The tachyonus can cause acute infection of the host, and the slow colonies in the capsule are the main forms of the chronic infection. The mechanism of the mutual transformation between the tachyonus and the gonogoniosis is unclear. There is no special drug for the treatment of toxoplasmosis before the eye. Drugs such as ethamimidine and acetyl spiramycin are the main drugs, these drugs kill and inhibit the tachyonus, but have little effect on the retardant in the capsule. But the activation of the retarded colonies in the capsule can cause great harm to the host. Therefore, the study of the mutual conversion mechanism between the tachyonus and the retardant of the Toxoplasma gondii has become the research of the toxoplasmosis. One of the hotspots and difficulties in the study.
In recent years, the development of reverse genetics based on RNA interference and transgenic technology has provided us with a method to analyze gene function from gene change. This study synthesizes the current research status of the mutual transformation of Toxoplasma gondii and retarded colonies and the main problems in the study, using Transgenesis, RNAi and Reverse genetics. The principle and method of the cell model of the mutual transformation between the tachyonus RH of Toxoplasma gondii and the retarded colonies in vitro, clone the coding sequence of the early differential gene and construct the reverse repeat sequence RNAi vector driven by the heat shock protein 70 (HSP70) gene promoter of Toxoplasma gondii, and build a set of Reverse Genet for the analysis of the gene function of Toxoplasma gondii. The ICs technology platform was used to analyze the genes that the tachyonus began to express early in the transformation of the retarded colonies to clarify the function and function of these molecules in the transformation of the tachyonite to the retarded colonies.
1. Establishment of an in vitro transformation model between tachyzoites and bradyzoites of Toxoplasma gondii
The mice intraperitoneally inoculated the tachyonus RH of Toxoplasma gondii, after 3-4 days, the mouse peritoneal fluid was collected. The host cells were released from the host cells of the domestic 4.5 needle syringes repeatedly sucking the sucking body suspension. The parasite suspension was filtered by the filter membrane with the diameter of 3 micron m. The purity of the tachyonus could reach more than 98%.
The purified Toxoplasma gondii RH strain tachyonus was inoculated in the NIH3T3 cell monolayer (NIH3T3 cell monolayer growth reached 60%-70%, inoculated tachyonus, culture medium containing 5% newborn bovine serum), cultured in 37 centigrade, 5%CO_2 culture box, rapid proliferation of tachyonite, massive destruction of host cells, and culturing Toxoplasma gondii in 5-6 days. A tachyonus, flask culture or experimental study.
In order to explore the induction and transformation of tachyonus from Toxoplasma gondii to retarded colonies under alkaline conditions, RH strain tachyonus was inoculated NIH3T3 cell monolayer according to the proportion of 1:10. After inoculation of 4h, the cell monolayer was gently flushed with pH8.1 DMEM medium to remove the Toxoplasma gondii tachyonus from the non invasive cells, and then the DMEM medium of pH8.1 (containing 2.5% newborn bovine serum) was placed in 3. The circular cystic corpuscles in the cytoplasm of NIH3T3 cells were observed under the 96h. microscope at 7 C, and the cysts of different sizes were found in the cytoplasm of the cells. The cyst wall was highly refracted. The total RNA was extracted and cultured. The method of RT-PCR was used to detect the retarded antigen 1 (BAG1) mRNA of the slow colonization antigen, and the 700bp target bands were obtained, and the PCR products were cloned to T. After the sequencing, the homology of the published BAG1 gene mRNA was 99.3%, and the sequence of the DNA intron was no genome. The results showed that the formation of the retarded colonies was successfully induced. In order to monitor the transformation process of the tachyonus to the retarded colonies, a bottle of the cultured insect body was extracted, the total RNA was extracted, and the Toxoplasma beta microtubule eggs were extracted from the induction of the 0,24,48,72,96 hour. The mRNA RT - PCR product of white was used as a reference to amplify the BAG1 gene mRNA by semi quantitative RT PCR. The result showed that the transcription level of BAG1 mRNA increased gradually with the prolongation of induction time, indicating that more Toxoplasma tachycolonies were converted to retarded colonies with the extension of induction time. To induce the transformation of the slow colonies to the tachyonite, the pH8.1 alkaline strip was used. The pH7.2 medium and normal culture conditions were restored by the induced colonies. After 24h, the tachyonus of the Toxoplasma gondii was found in the solution. After 2-3 days, the tachyonus began to proliferate. It was necessary to supplement the NIH3T3 cells to continue the culture.
In order to explore the effect of heat shock on the transformation of Toxoplasma gondii to the retarded colonies, the conventional cultured NIH3T3 cells were put into 37, 39, 41, 43, and 5%CO_2 culture incubator for 3h, and the purified RH tachyonus was inoculated in proportion to the 1:10 proportion. After inoculation of 4h, the cell monolayer was gently flushed with the corresponding temperature preheated DMEM medium. After intruding the tachyonus of Toxoplasma gondii, they were placed at 37, 39, 41, 43, respectively, and cultured in 5%CO_2 incubator for 48h. The culture mixture was collected and the total RNA was extracted. The results of RT-RCR. showed that the tachyonite could not be induced to form a retarder under the condition of 39 degrees centigrade. The induction condition of the tachyonite was.43 centigrade for the growth condition of the tachyonite. Under the guidance conditions, NIH3T3 host cells are difficult to grow, and can not provide a stable intracellular environment for Toxoplasma tachygonite, which is not conducive to the transformation of Toxoplasma RH tachyonus to the retarded colonies at.41 C, and the degeneration and necrosis of the host cells of the host cell are relatively slow, and can provide a relatively stable intracellular environment for the Toxoplasma tachyonus, and the Toxoplasma gondii The tachyonus of RH strain can be induced to be retarded.
2. Construction of a Genetically and Inducible RNAi Vector System of Toxoplasma gondii
The construction of a green fluorescent protein gene carrier driven by the main surface antigen 1 (SAG1) gene promoter of Toxoplasma gondii: the purpose of building the vector is one of the fluorescent screening markers of the transgenic Toxoplasma gondii, on the other hand, as a transgenic Toxoplasma gondii is a tachyonite or a slow colonization, or a intermediate state between the two. Primers were designed to amplify the SAG1 gene 5'UTR promoter sequence (SAG1 / 5UTR), SAG1 gene 3'UTR sequence (SAG1 / 3UTR) and green fluorescent protein coding sequence (eGFP) respectively, and the green fluorescent protein gene carrier driven by the promoter of Toxoplasma gondii SAG1 gene promoter was constructed by PCR. P), the result of sequence determination is correct.
2. toxoplasmosis inducible reverse repeat sequence RNAi vector construction: primers were designed to amplify the 5'UTR promoter sequence (HSP70 / 5UTR) of Toxoplasma gondii HSP70 gene (HSP70 / 5UTR), HSP70 gene 3'UTR sequence (HSP70 / 3UTR) and beta microtubulin gene intron C sequence (IntronC), and the Toxoplasma gondii heat shock protein was constructed by enzyme digestion. The gene promoter was driven by using the intron C sequence of the beta tubulin gene as the interval sequence and the HSP70 gene 3'UTR sequence as the reverse repeat sequence RNAi vector pBSK-HSP70 / 5UTR-IntronC-HSP70 / 3UTR for the transcription termination signal, and the sequencing results were correct.
3. Toxoplasma gondii can be inherited and inducible RNAi vector system construction: the carrier pHANA-0.5 has the function of the Toxoplasma gondii shuttle carrier, and the SAG1 / 5UTR-eGFP-SAG1 / 3UTR fragment in the carrier pBSK-SAG1 / GFP is cloned into the carrier pBSK-HSP70 / 5UTR-IntronC-HSP70 / 3UTR to form carrier pBSK-GFP-Hairpin, and then the GFP-Ha in the carrier The irpin fragment was cloned into the vector pHANA-0.5 to form the inheritable and inducible RNAi vector system pHANA-hairpin of Toxoplasma gondii. The sequencing results were correct.
4. SAG1 gene RNAi vector of Toxoplasma gondii: to RNAi the SAG1 gene of Toxoplasma gondii, design primers, amplify the positive and reverse sequence of SAG1 gene by PCR, and clone the positive and reverse sequences to the corresponding enzyme tangent site of the carrier pHANA-hairpin through PCR, and construct the pHANA-hairpin / SAG1, sequence determination of the RNAi carrier of the SAG1 gene. The result is correct.
5. BAGl gene RNAi vector of Toxoplasma gondii: to RNAi the BAG1 gene of Toxoplasma gondii, design primers, amplify the positive and reverse sequence of BAG1 gene by PCR, and clone the positive and reverse sequences to the corresponding enzyme tangent site of the carrier pHANA-hairpin through PCR, and construct the pHANA-hairpin / BAG1, sequence determination of the RNAi carrier of the BAG1 gene. The result is correct.
Establishment of transgenic Toxoplasma gondii strains and preliminary study on RNAi
Two transgenic Toxoplasma gondii strains were obtained by converting plasmids pHANA-hairpin / SAG1 and pHANA-hairpin / BAG1 to Toxoplasma gondii tachygonite by electrical transformation. The tachygonite and plasmid DNA 10 u g were suspended in the 800 Mug shock buffer liquid cytomix. The voltage 0.4kV in the 4mm gap electroporation cup, the capacitance of 800 mu 1 times, and the time delay. After 24h transfection of Toxoplasma gondii transfected with 6-9msec., the expression of green fluorescence was observed in two strains of transgenic Toxoplasma gondii, and two strains of transgenic Toxoplasma gondii tachygonite total RNA were extracted, and RT-PCR was carried out respectively. The results showed that the transcription of the GFP gene could be detected and the plasmid was successfully introduced into the tachygonite. The expression of two strains of transgenic Toxoplasma gondii was induced by alkaline environment (pH8.1). After induction and culture of 96h, green fluorescence was observed under the fluorescence inversion microscope, and two strains of transgenic Toxoplasma gondii induced total RNA and RT-PCR were extracted respectively, and all of them could detect SAG1, BAG1 and GFP base. The possible causes of this phenomenon are as follows: 1. parts of the transgenic Toxoplasma gondii have not been transformed into the retarded colonies; the 2. transformation of the transgenic Toxoplasma gondii is in the middle state of a tachyonite and the retarded colonies, and does not form mature retarded colonies and cysts; 3. induced interference with the SAG1 gene or BAG1 gene may be a tachyonite. Transgenic Toxoplasma gondii is still in tachyzoite state because of the influence of the transformation process to bradyzoites. These problems need further study.
【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R383
【引证文献】
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1 杨欢欢;弓形虫SAG1转基因蜥蜴利什曼原虫的构建及鉴定[D];吉林农业大学;2011年
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