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  本文選題：斯氏艾美耳球蟲　切入點：病毒　出處：《吉林大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：兔球蟲病是由艾美耳屬球蟲引起的寄生性原蟲病。能感染家兔的球蟲共有17種，其中斯氏艾美耳球蟲（Eimeria stiedai）是寄生在膽管上皮細胞內(nèi)，因其致病性最強及寄生部位的特殊性，而引起了學(xué)者的普遍關(guān)注。其分子生物學(xué)和細胞生物學(xué)特性了解不夠清楚，導(dǎo)致尚無有效的防治方法。球蟲病毒的發(fā)現(xiàn)為球蟲的特性研究提供了新的方向。斯氏艾美耳球蟲病毒樣粒子早在1989年就有報道[1]，但在當(dāng)時并未受到重視，只是觀察到了病毒樣粒子存在，并未對其進行進一步的研究，也并未在分類學(xué)上有正式的歸屬。目前在國內(nèi)此方面的研究尚屬空白。本試驗對斯氏艾美耳球蟲卵囊內(nèi)病毒樣粒子進行研究，為球蟲病的有效防治及斯氏艾美耳球蟲的細胞和分子生物學(xué)特性等方面的探究提供新的思路。攜病毒斯氏艾美耳球蟲蟲株的分離篩選： 本研究采用臨床收集兔球蟲卵囊經(jīng)人工感染幼兔，從肝臟中分離純化得到一株斯氏艾美耳球蟲球蟲株。通過光學(xué)顯微境觀察其卵囊平均大小為21.5μm×19.2μm，壁光滑，無卵囊殘體有四個孢子囊，均呈長橢圓形。進一步經(jīng)RT-PCR鑒定，說明該球蟲蟲株為斯氏艾美耳球蟲蟲株。通過提取其孢子化卵囊的總核酸進行瓊脂糖電泳分析，發(fā)現(xiàn)除該球蟲基因組外還存在一條6.5kb的核酸帶，初步鑒定該蟲株為攜病毒斯氏艾美耳球蟲蟲株。斯氏艾美耳球蟲病毒的鑒定 本試驗通過對其進行核酸酶的敏感性試驗及高鹽(0.3M NaCl)保護性試驗證明該病毒的遺傳物質(zhì)為雙鏈RNA（dsRNA）。通過采用α-32P-UTP摻入法檢測到斯氏艾美耳球蟲病毒樣顆粒具有RNA依賴性RNA聚合酶（RDRP）活性。通過對該球蟲孢子化卵囊粗提液進行蔗糖密度梯度離心及電鏡觀察到斯氏艾美耳球蟲病毒樣粒子呈球形，二十面體對稱結(jié)構(gòu)，無囊膜，直徑為35nm。斯氏艾美耳球蟲病毒基因組部分cDNA序列測定 本試驗通過DNA酶消化DNA及利用高鹽保護原理在高鹽緩沖液中用RNaseA對單鏈RNA進行消化以達到純化dsRNA的目的。以純化后的dsRNA為模板，錨定隨機引物為引物進行反轉(zhuǎn)錄，以該錨定序列為引物，進行單引物PCR。將PCR回收產(chǎn)物連接到pMD-18T載體上進行測序。測序結(jié)果經(jīng)BLAST X在線比對，結(jié)果顯示有763bp與鶉雞皰疹病毒2型（Gallid herpesvirus2）編碼衣殼蛋白的基因同源率為40.8%，，推測為斯氏艾美耳球蟲病毒基因組中編碼衣殼的核酸序列。
[Abstract]:Rabbit coccidiosis is a parasitic protozoa caused by Eimeria. There are 17 species of coccidiosis that can infect rabbits. Eimeria stiedaii (Eimeria stiedaii) is parasitic in the epithelial cells of bile duct, because of its strong pathogenicity and the particularity of the parasitic site. It has aroused widespread concern among scholars. Its molecular and cellular biological characteristics are not well understood. The discovery of the coccidia virus provides a new direction for the study of the characteristics of coccidiosis. The presence of virus-like particles was observed without further study. There is no formal taxonomic attribution. At present, the research in this field is still blank. In this study, the viriform particles in oocysts of Eimeria skrjabini were studied. To provide a new idea for the effective control of coccidiosis and the study of cell and molecular biological characteristics of Eimeria spp. In this study, a strain of Eimeria skrjabini coccidia was isolated and purified from the liver by clinical collection of rabbit coccidia oocysts. The average size of the oocysts was 21.5 渭 m 脳 19.2 渭 m, and the wall was smooth. There were four sporangial spore sacs in the absence of oocysts, all of which were long oval. Further identified by RT-PCR, the coccidia strain was Eimeria skrjabini strain. The total nucleic acid of sporulated oocysts was extracted and analyzed by agarose electrophoresis. A 6.5kb nucleic acid band was found in addition to the genome of the coccidia, and the strain was identified as a virus carrying Eimeria spp. In this experiment, the nuclease sensitivity test and the high salt 0.3M NaCl-protective test proved that the genetic material of the virus was double-stranded RNA-dsRNA. By using 偽 -32P-UTP incorporation method, it was found that the virus-like particles of Eimeria skrjabini had RNA isoform. By sucrose density gradient centrifugation and electron microscopy, the viriform particles of Eimeria skrjabini were observed to be spherical, by means of sucrose density gradient centrifugation and electron microscopy. Determination of partial cDNA sequence of the genome of Eimeria spp. Virus with a symmetrical structure of 20 hedrons, no capsule and a diameter of 35 nm. In this experiment, DNA was digested by DNA and RNaseA was used in high salt buffer to digest DNA. The purified dsRNA was used as template and anchored random primer was used as primer for reverse transcription. Using the anchored sequence as a primer, a single primer PCR.The PCR recovery product was linked to the pMD-18T vector for sequencing. The sequencing results were compared with BLAST X online. The results showed that the homology of 763 BP gene encoding capsid protein with Gallid herpesvirus2 of herpesvirus 2 was 40.8, which was deduced to be the nucleic acid sequence encoding capsid in the genome of Eimeria spp.
【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】：2012
【分類號】：R382.32

【參考文獻】

相關(guān)期刊論文 前2條

1 陳麗鳳;李建華;劉全;趙月平;曹利利;張西臣;;犬賈第蟲病毒轉(zhuǎn)染載體介導(dǎo)的綠色熒光蛋白在犬賈第蟲體內(nèi)的表達[J];中國寄生蟲學(xué)與寄生蟲病雜志;2007年01期

2 于詠蘭,汪明;隨機擴增多態(tài)性DNA技術(shù)對雞的3種艾美耳球蟲的鑒別[J];中國獸醫(yī)雜志;2000年08期

相關(guān)碩士學(xué)位論文 前5條

1 韓乾忠;柔嫩艾美耳球蟲病毒的鑒定及細胞內(nèi)分布[D];吉林大學(xué);2011年

2 刁玉梅;微小隱孢子蟲病毒衣殼蛋白單克隆抗體的制備及初步應(yīng)用[D];吉林大學(xué);2011年

3 郭瑞娟;陰道毛滴蟲病毒轉(zhuǎn)染載體的構(gòu)建及EGFP在陰道毛滴蟲內(nèi)的表達[D];吉林大學(xué);2007年

4 于新友;微小隱孢子蟲病毒轉(zhuǎn)染載體的構(gòu)建及GFP在其體內(nèi)的表達[D];吉林大學(xué);2007年

5 張建坤;陰道毛滴蟲核酶嵌合型病毒載體FRzS的構(gòu)建[D];吉林大學(xué);2009年

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