本文選題：旋毛蟲 + 期特異性基因　； 參考：《吉林大學(xué)》2007年碩士論文

【摘要】： 由于旋毛蟲存在三個不同的發(fā)育時期,而各個發(fā)育時期具有不同的抗原表達,因此研究旋毛蟲期特異性基因有助于探明旋毛蟲侵襲、致病機理以及包囊形成的分子機制。我們利用分子生物學(xué)和免疫學(xué)方法對本室業(yè)已發(fā)現(xiàn)的2條新生幼蟲期特異性(即旋毛蟲包囊形成時期)基因——II型脫氧核糖核酸酶基因T314和絲氨酸蛋白酶基因T668進行了轉(zhuǎn)錄及表達特性研究,并探討了其參與旋毛蟲寄生及包囊形成過程中的可能機制。 根據(jù)T314和T668 cDNA序列設(shè)計特異引物,從感染旋毛蟲的小鼠骨骼肌中提取總RNA,應(yīng)用RT-PCR技術(shù)分別反轉(zhuǎn)錄出T314和T668 cDNA,在分子水平上對這2個基因的轉(zhuǎn)錄時空進行了鑒定。結(jié)果顯示:T314、T668基因均從新生幼蟲出生即開始轉(zhuǎn)錄,隨著日齡的增加mRNA的量逐漸減少,T314 mRNA在感染后第20天仍能檢測到,而T668 mRNA于感染后11-14天轉(zhuǎn)錄趨于停止。 分別利用本室制備的兔抗T314和T668重組蛋白抗體,采用免疫組化技術(shù),觀察T314和T668基因編碼蛋白的組織學(xué)定位。結(jié)果表明:T314、T668蛋白在新生幼蟲感染后第1天均有表達,T314蛋白分布于蟲體表皮,而T668蛋白分布于蟲體的桿狀體區(qū)域;T314蛋白在感染后7-20天分泌到蟲體外的保姆細(xì)胞細(xì)胞質(zhì)上,而T668蛋白則在感染后6-13天分泌到蟲體外腫大的肌細(xì)胞核上。該結(jié)果預(yù)示著:T314蛋白(即DNase II)可能與旋毛蟲寄生所需要厭氧環(huán)境的形成有關(guān),可能的機制為DNase II降解線粒體DNA,使包囊內(nèi)的有氧代謝轉(zhuǎn)變?yōu)闊o氧代謝;而T668蛋白(即絲氨酸蛋白酶)可能參與包囊的形成,其可能的機制是作為轉(zhuǎn)錄因子調(diào)控腫大的肌細(xì)胞核大量表達膠原蛋白從而形成包囊。 從pMD-18T-T314重組質(zhì)粒中擴增T314目的片段,重組到PCR II載體中。利用SP6和T7 RNA聚合酶體外轉(zhuǎn)錄合成siRNA。通過浸泡將siRNA導(dǎo)入新生幼蟲體內(nèi)進行T314基因沉默,發(fā)現(xiàn)T314基因沉默后蟲體總數(shù)減少,形成的包囊大部分空泡化,初步驗證T314基因可能與旋毛蟲包囊形成無關(guān)。
[Abstract]:Since there are three different developmental stages of Trichinella spiralis, and different antigens are expressed in each developmental stage, studying the specific genes of Trichinella spiralis is helpful to understand the invasion, pathogenesis and the molecular mechanism of cyst formation of Trichinella spiralis. We have used molecular biology and immunological methods to identify two new larval stage specific genes (I. e., the stage of cysts formation of Trichinella spiralis): type II deoxyribonuclease gene T314 and serine protease gene. The transcriptional and expression characteristics of T668 were studied. The possible mechanism of its involvement in parasitism and cyst formation of Trichinella spiralis was also discussed. According to the sequence of T314 and T668 cDNA, specific primers were designed to extract total RNAs from skeletal muscle of mice infected with Trichinella spiralis. T314 and T668 cDNAwere inverted by RT-PCR technique. The transcriptional time and space of these two genes were identified at molecular level. The results showed that the T314T668 gene was transcribed from the birth of the newborn larva. With the increase of age, the amount of T314 mRNA could be detected on the 20th day after infection, while the transcription of T668 mRNA tended to stop at 11-14 days after infection. Using rabbit anti-T314 and T668 recombinant protein antibodies prepared in our laboratory, the histological localization of T314 and T668 gene encoded proteins was observed by immunohistochemical technique. The results showed that T314T668 protein was expressed in the epidermis of the larva on the first day after infection, while the T668 protein was secreted into the cytoplasm of the nanny in vitro 7-20 days after infection. The T 668 protein was secreted 6-13 days after infection to the swollen muscle nucleus of the insect in vitro. The results suggest that the DNase II protein may be related to the formation of anaerobic environment required by Trichinella spiralis parasitism. The possible mechanism is that DNase II degrades mitochondrial DNA and changes aerobic metabolism into anaerobic metabolism in the cyst. T668 protein (serine protease) may be involved in the formation of cysts, and its possible mechanism is to regulate the expression of collagen protein in the enlarged muscle nucleus as a transcription factor. The target fragment of T314 was amplified from pMD-18T-T314 recombinant plasmid and recombined into PCR II vector. SiRNA was synthesized by in vitro transcription of SP6 and T7 RNA polymerase. SiRNA was introduced into the newborn larva for T314 gene silencing. It was found that the total number of body decreased and most of the cysts were vacuolated after the silencing of T314 gene. It was preliminarily verified that T314 gene may not be related to the formation of trichinella cyst.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R383;S852.7

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