斯氏艾美耳球虫病毒的鉴定及特性研究

发布时间：2018-03-15 20:52

本文选题：斯氏艾美耳球虫　切入点：病毒　出处：《吉林大学》2012年硕士论文　论文类型：学位论文

【摘要】：兔球虫病是由艾美耳属球虫引起的寄生性原虫病。能感染家兔的球虫共有17种，其中斯氏艾美耳球虫（Eimeria stiedai）是寄生在胆管上皮细胞内，因其致病性最强及寄生部位的特殊性，而引起了学者的普遍关注。其分子生物学和细胞生物学特性了解不够清楚，导致尚无有效的防治方法。球虫病毒的发现为球虫的特性研究提供了新的方向。斯氏艾美耳球虫病毒样粒子早在1989年就有报道[1]，但在当时并未受到重视，只是观察到了病毒样粒子存在，并未对其进行进一步的研究，也并未在分类学上有正式的归属。目前在国内此方面的研究尚属空白。本试验对斯氏艾美耳球虫卵囊内病毒样粒子进行研究，为球虫病的有效防治及斯氏艾美耳球虫的细胞和分子生物学特性等方面的探究提供新的思路。携病毒斯氏艾美耳球虫虫株的分离筛选：本研究采用临床收集兔球虫卵囊经人工感染幼兔，从肝脏中分离纯化得到一株斯氏艾美耳球虫球虫株。通过光学显微境观察其卵囊平均大小为21.5μm×19.2μm，壁光滑，无卵囊残体有四个孢子囊，均呈长椭圆形。进一步经RT-PCR鉴定，说明该球虫虫株为斯氏艾美耳球虫虫株。通过提取其孢子化卵囊的总核酸进行琼脂糖电泳分析，发现除该球虫基因组外还存在一条6.5kb的核酸带，初步鉴定该虫株为携病毒斯氏艾美耳球虫虫株。斯氏艾美耳球虫病毒的鉴定本试验通过对其进行核酸酶的敏感性试验及高盐(0.3M NaCl)保护性试验证明该病毒的遗传物质为双链RNA（dsRNA）。通过采用α-32P-UTP掺入法检测到斯氏艾美耳球虫病毒样颗粒具有RNA依赖性RNA聚合酶（RDRP）活性。通过对该球虫孢子化卵囊粗提液进行蔗糖密度梯度离心及电镜观察到斯氏艾美耳球虫病毒样粒子呈球形，二十面体对称结构，无囊膜，直径为35nm。斯氏艾美耳球虫病毒基因组部分cDNA序列测定本试验通过DNA酶消化DNA及利用高盐保护原理在高盐缓冲液中用RNaseA对单链RNA进行消化以达到纯化dsRNA的目的。以纯化后的dsRNA为模板，锚定随机引物为引物进行反转录，以该锚定序列为引物，进行单引物PCR。将PCR回收产物连接到pMD-18T载体上进行测序。测序结果经BLAST X在线比对，结果显示有763bp与鹑鸡疱疹病毒2型（Gallid herpesvirus2）编码衣壳蛋白的基因同源率为40.8%，，推测为斯氏艾美耳球虫病毒基因组中编码衣壳的核酸序列。
[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 伪 -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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R382.32

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