【摘要】：剛地弓形蟲(Toxoplasma gondii)是一種呈世界性分布的專性細(xì)胞內(nèi)寄生原蟲，可寄生在除紅細(xì)胞外的幾乎所有有核細(xì)胞中，引起人獸共患的弓形蟲病。弓形蟲是一種機(jī)會(huì)性致病原蟲，機(jī)體免疫功能正常者感染弓形蟲一般沒有明顯癥狀，為隱性感染狀態(tài)。但對于免疫功能受損或缺陷者，如腫瘤病人，AIDS患者等，弓形蟲在患者體內(nèi)可以大量增殖，引起全身播散性損害，嚴(yán)重者可導(dǎo)致死亡。此外，妊娠期感染弓形蟲可通過胎盤垂直傳播，引起早產(chǎn)、流產(chǎn)、死胎、畸胎或嬰兒發(fā)育畸形等。近年來，，由于艾滋病的廣泛流行和寵物飼養(yǎng)的逐漸增多，弓形蟲的危害也日益突出，弓形蟲感染成為一個(gè)嚴(yán)重的公共衛(wèi)生問題。 弓形蟲速殖子與緩殖子的相互轉(zhuǎn)化，是弓形蟲致病的中心環(huán)節(jié)。速殖子可引起宿主的急性感染，而包囊內(nèi)的緩殖子是形成慢性感染的主要形式，弓形蟲速殖子與緩殖子相互轉(zhuǎn)化的機(jī)制尚不清楚。目前弓形蟲病的治療尚無特效藥物，主要以乙胺嘧啶，乙酰螺旋霉素等藥物為主，這些藥物對速殖子有殺滅、抑制作用，而對包囊內(nèi)的緩殖子幾乎沒有作用。但包囊內(nèi)緩殖子的活化，能對宿主造成極大的危害。因此，探討弓形蟲速殖子與緩殖子相互轉(zhuǎn)換機(jī)制的研究成為弓形蟲病研究的熱點(diǎn)和難點(diǎn)之一。 近年來，以RNA干擾和轉(zhuǎn)基因技術(shù)為基礎(chǔ)的反向遺傳學(xué)的發(fā)展，為我們提供了一個(gè)從基因變化分析基因功能的方法。本研究綜合目前弓形蟲速殖子與緩殖子相互轉(zhuǎn)化的研究現(xiàn)狀及研究中存在的主要問題，采用Transgenesis、RNAi和Reverse genetics的原理和方法，建立弓形蟲RH株速殖子與緩殖子體外相互轉(zhuǎn)化的細(xì)胞模型，克隆轉(zhuǎn)化早期差顯基因的編碼序列，構(gòu)建弓形蟲熱休克蛋白70(HSP70)基因啟動(dòng)子驅(qū)動(dòng)的反向重復(fù)序列RNAi載體系統(tǒng)，建立一套進(jìn)行弓形蟲基因功能分析的Reverse Genetics技術(shù)平臺(tái)，并選取在速殖子向緩殖子轉(zhuǎn)化早期開始表達(dá)的基因進(jìn)行重點(diǎn)分析，以闡明這些分子在速殖子向緩殖子轉(zhuǎn)化過程中的功能與作用。 一、弓形蟲體外速殖子與緩殖子相互轉(zhuǎn)化模型的建立 小鼠腹腔接種弓形蟲RH株速殖子，3-4天后收集小鼠腹腔液，用國產(chǎn)4.5號(hào)針頭注射器反復(fù)抽吸蟲體懸液破裂宿主細(xì)胞釋放蟲體，蟲體懸液用孔徑3μm的濾膜過濾純化速殖子，速殖子純度可達(dá)98％以上。 將純化的弓形蟲RH株速殖子按照1：10的比例，接種于NIH3T3細(xì)胞單層(NIH3T3細(xì)胞單層生長達(dá)到60％-70％時(shí)接種速殖子，培養(yǎng)基含5％新生牛血清)，置于37℃，5％CO_2的培養(yǎng)箱中培養(yǎng)，速殖子增殖迅速，大量破壞宿主細(xì)胞，培養(yǎng)5-6天后收集弓形蟲速殖子，進(jìn)行分瓶培養(yǎng)或?qū)嶒?yàn)研究。 為探索堿性條件下弓形蟲速殖子向緩殖子的誘導(dǎo)轉(zhuǎn)化，按照1：10比例將RH株速殖子接種NIH3T3細(xì)胞單層，接種4h后，用pH8.1的DMEM培養(yǎng)基輕輕沖洗細(xì)胞單層以移去未侵入細(xì)胞的弓形蟲速殖子，再加入pH8.1的DMEM培養(yǎng)基(含2.5％新生牛血清)置于37℃空氣中培養(yǎng)96h。鏡下觀察可見NIH3T3細(xì)胞胞漿中出現(xiàn)大小不等的圓形囊性小體，囊壁折光性較強(qiáng)。收集培養(yǎng)蟲體提取總RNA，采用RT-PCR的方法進(jìn)行緩殖子期特異抗原緩殖子抗原1(BAG1)mRNA的檢測，結(jié)果得到700bp左右目的條帶，PCR產(chǎn)物克隆到T載體后測序，測序結(jié)果與已發(fā)表的BAG1基因mRNA同源性高達(dá)99.3％，無基因組DNA內(nèi)含子序列，結(jié)果表明成功誘導(dǎo)緩殖子的形成。為監(jiān)測速殖子向緩殖子轉(zhuǎn)化進(jìn)程，在誘導(dǎo)第0、24、48、72、96小時(shí)，分別取出一瓶培養(yǎng)蟲體，提取總RNA，以弓形蟲β—微管蛋白的mRNA RT—PCR產(chǎn)物為參照，進(jìn)行半定量RT—PCR擴(kuò)增BAG1基因mRNA，結(jié)果顯示，隨著誘導(dǎo)時(shí)間的延長，BAG1 mRNA的轉(zhuǎn)錄水平逐漸提高，表明隨著誘導(dǎo)時(shí)間的延伸，有更多的弓形蟲速殖子轉(zhuǎn)化成緩殖子。為誘導(dǎo)緩殖子向速殖子的轉(zhuǎn)化，將pH8.1堿性條件誘導(dǎo)形成的緩殖子恢復(fù)pH7.2培養(yǎng)基及正常培養(yǎng)條件，24h后可見溶液中出現(xiàn)游離弓形蟲速殖子，2-3天后速殖子開始大量增殖，需適當(dāng)補(bǔ)充NIH3T3細(xì)胞才能繼續(xù)培養(yǎng)。 為探索熱休克對弓形蟲速殖子向緩殖子的轉(zhuǎn)化影響，將常規(guī)培養(yǎng)的NIH3T3細(xì)胞分別放入37℃，39℃，41℃，43℃，5％CO_2培養(yǎng)箱中培養(yǎng)3h后，按照1：10比例接種純化的RH株速殖子，接種4h后，用相應(yīng)溫度預(yù)熱的DMEM培養(yǎng)基輕輕沖洗細(xì)胞單層，以移去未侵入細(xì)胞的弓形蟲速殖子后，分別放置在37℃，39℃，41℃，43℃，5％CO_2培養(yǎng)箱中培養(yǎng)48h，收集培養(yǎng)混合物，提取總RNA，進(jìn)行RT-RCR。結(jié)果表明39℃誘導(dǎo)條件下，速殖子增殖較快，不能誘導(dǎo)形成緩殖子，39℃誘導(dǎo)條件還是適于速殖子的生長條件。43℃誘導(dǎo)條件下，NIH3T3宿主細(xì)胞難以生長，不能為弓形蟲速殖子提供一個(gè)穩(wěn)定的細(xì)胞內(nèi)環(huán)境，不利于弓形蟲RH株速殖子向緩殖子的轉(zhuǎn)化。41℃誘導(dǎo)條件下，NIH3T3宿主細(xì)胞變性壞死相對較慢，能為弓形蟲速殖子提供一個(gè)相對穩(wěn)定的細(xì)胞內(nèi)環(huán)境，弓形蟲RH株速殖子能誘導(dǎo)轉(zhuǎn)化為緩殖子。 二、弓形蟲可遺傳及可誘導(dǎo)的RNAi載體系統(tǒng)的構(gòu)建 1．弓形蟲主要表面抗原1(SAG1)基因啟動(dòng)子驅(qū)動(dòng)的綠色熒光蛋白基因載體的構(gòu)建：構(gòu)建該載體的目的是一方面作為轉(zhuǎn)基因弓形蟲的一種熒光篩選標(biāo)志，另一方面作為轉(zhuǎn)基因弓形蟲是處于速殖子或緩殖子，還是兩者之間中間狀態(tài)的一種指示。設(shè)計(jì)引物，通過PCR分別擴(kuò)增SAG1基因5'UTR啟動(dòng)子序列(SAG1／5UTR)、SAG1基因3'UTR序列(SAG1／3UTR)及綠色熒光蛋白編碼序列(eGFP)，通過酶切連接，構(gòu)建弓形蟲SAG1基因啟動(dòng)子驅(qū)動(dòng)的綠色熒光蛋白基因載體pBSK-SAG1／5UTR-eGFP-SAG1／3UTR(pBSK-SAG1／GFP)，序列測定結(jié)果正確。 2．弓形蟲可誘導(dǎo)的反向重復(fù)序列RNAi載體的構(gòu)建：設(shè)計(jì)引物，通過PCR分別擴(kuò)增弓形蟲HSP70基因5'UTR啟動(dòng)子序列(HSP70／5UTR)、HSP70基因3'UTR序列(HSP70／3UTR)及β-微管蛋白基因內(nèi)含子C序列(IntronC)，通過酶切連接，構(gòu)建以弓形蟲熱休克蛋白HSP70基因啟動(dòng)子進(jìn)行驅(qū)動(dòng)的，以β-微管蛋白基因內(nèi)含子C序列作為間隔序列，以HSP70基因3'UTR序列作為轉(zhuǎn)錄終止信號(hào)的反向重復(fù)序列RNAi載體pBSK-HSP70／5UTR-IntronC-HSP70／3UTR，序列測定結(jié)果正確。 3．弓形蟲可遺傳及可誘導(dǎo)的RNAi載體系統(tǒng)的構(gòu)建：利用載體pHANA-0.5具有弓形蟲穿梭載體的功能，將載體pBSK-SAG1／GFP中的SAG1／5UTR-eGFP-SAG1／3UTR片段克隆到載體pBSK-HSP70／5UTR-IntronC-HSP70／3UTR中形成載體pBSK-GFP-Hairpin，再將該載體中的GFP-Hairpin片段克隆到載體pHANA-0.5中形成弓形蟲可遺傳及可誘導(dǎo)的RNAi載體系統(tǒng)pHANA-hairpin，序列測定結(jié)果正確。 4．弓形蟲SAG1基因RNAi載體的構(gòu)建：為對弓形蟲SAG1基因進(jìn)行RNAi，設(shè)計(jì)引物，通過PCR分別擴(kuò)增SAG1基因的正向和反向序列，通過酶切連接，將正向和反向序列克隆到載體pHANA-hairpin相應(yīng)酶切位點(diǎn)，構(gòu)建SAG1基因RNAi載體pHANA-hairpin／SAG1，序列測定結(jié)果正確。 5．弓形蟲BAGl基因RNAi載體的構(gòu)建：為對弓形蟲BAG1基因進(jìn)行RNAi，設(shè)計(jì)引物，通過PCR分別擴(kuò)增BAG1基因的正向和反向序列，通過酶切連接，將正向和反向序列克隆到載體pHANA-hairpin相應(yīng)酶切位點(diǎn)，構(gòu)建BAG1基因RNAi載體pHANA-hairpin／BAG1，序列測定結(jié)果正確。 三、轉(zhuǎn)基因弓形蟲品系的建立及RNAi初步研究 分別將質(zhì)粒pHANA-hairpin／SAG1和pHANA-hairpin／BAG1通過電轉(zhuǎn)化導(dǎo)入弓形蟲RH株速殖子獲得兩個(gè)轉(zhuǎn)基因弓形蟲品系。將1X10~7弓形蟲速殖子和質(zhì)粒DNA 10μg重懸于800μl電擊緩沖液cytomix中。在4mm間隙電穿孔杯中以電壓0.4kv，電容800μF電擊1次，時(shí)間延遲為6-9msec。轉(zhuǎn)染弓形蟲培養(yǎng)24h后，在熒光倒置顯微鏡下觀察，兩個(gè)品系轉(zhuǎn)基因弓形蟲均可觀察到綠色熒光的表達(dá)，分別提取兩個(gè)品系轉(zhuǎn)基因弓形蟲速殖子總RNA，進(jìn)行RT-PCR，結(jié)果均能檢測到GFP基因的轉(zhuǎn)錄，證明質(zhì)粒成功導(dǎo)入弓形蟲速殖子并進(jìn)行了表達(dá)。將兩個(gè)品系轉(zhuǎn)基因弓形蟲速殖子進(jìn)行堿性環(huán)境(pH8．1)誘導(dǎo)，誘導(dǎo)培養(yǎng)96h后，在熒光倒置顯微鏡下觀察，均可觀測到有綠色熒光表達(dá)，分別提取兩個(gè)品系轉(zhuǎn)基因弓形蟲誘導(dǎo)后蟲體總RNA，進(jìn)行RT-PCR，結(jié)果均能檢測到SAG1、BAG1及GFP基因的轉(zhuǎn)錄。出現(xiàn)這種現(xiàn)象可能的原因有：1．部分轉(zhuǎn)基因弓形蟲速殖子未轉(zhuǎn)化為緩殖子；2．部分轉(zhuǎn)化的轉(zhuǎn)基因弓形蟲處于一種速殖子與緩殖子的中間狀態(tài)，未形成成熟的緩殖子和包囊；3．誘導(dǎo)干擾的SAG1基因或BAG1基因可能對速殖子向緩殖子轉(zhuǎn)化過程有影響，使得轉(zhuǎn)基因弓形蟲仍處于速殖子狀態(tài)，這些問題尚需進(jìn)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.
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R383

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 楊歡歡;弓形蟲SAG1轉(zhuǎn)基因蜥蜴利什曼原蟲的構(gòu)建及鑒定[D];吉林農(nóng)業(yè)大學(xué);2011年

2 李博;表達(dá)弓形蟲ROP18基因重組蜥蜴利什曼原蟲的構(gòu)建及鑒定[D];吉林農(nóng)業(yè)大學(xué);2012年

本文編號(hào)：2144097