【作者】 王玉洁；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2004， 硕士

【摘要】 猪传染性胃肠炎病毒(Transmissible Gastroenteritis Virus, TGEV)属于冠状病毒科冠状病毒属，其可导致猪传染性胃肠炎。对养猪业危害性极大。抗毒素和抗生素等常规药物对本病无任何治疗作用，预防该病的有效方法是疫苗接种。本研究以猪传染性胃肠炎病毒感染的乳猪小肠病料为材料，自行设计两对引物，通过套式（RT-nested）PCR及普通的RT-PCR扩增出猪传染性胃肠炎病毒M基因全序列，经测序、同源性比较后，进行深入的分析，主要研究结果如下： 1．本试验以乳猪小肠组织病料为材料，用Trizol法提取总RNA，经甲醛凝胶变性电泳，能清晰的看到28s、18srRNA两条亮带以及一条较弱的5srRNA带。结果表明，所提取的RNA完整性好，没有发生降解，完全可以用于RT-PCR反应。 2．根据GeneBank中发表的猪传染性胃肠炎病毒M基因序列，利用软件设计两对引物（外引物和内引物），在普通的RT- PCR方法基础上，建立了 RT-nested PCR体系，扩增出猪传染性胃肠炎病毒M基因的全序列，使用内外引物扩增的片段长度分别为1037bp、1328bp 。结果表明，用双引物的RT-nested PCR较普通的RT- PCR扩增方法更具特异性，重复性好，能很好的避免病毒检测时假阳性的出现。且较传统的方法如电镜技术、免疫荧光技术、ELISA等更方便可靠，完全可以作为一种新型的、特异性强的病毒检测的方法。 3．利用 RT- PCR 方法，扩增出猪传染性胃肠炎病毒 M 基因全序列，长度为 1 328 bp，扩增产物经回收、转化,克隆在 pGEM-T Easy 载体的 T 位点上。用 EcoRⅠ酶切鉴定重组质粒 DNA，挑选出阳性克隆菌，进行序列测定后。利用 CLUSTAL（1.82）软件进行同源性分析。结果显示，扩增的猪传染性胃肠炎病毒 M 基因序列与 GeneBank 中登录号为AY587883 和 AF302262 的猪传染性胃肠炎病毒 M 基因的核苷酸序列同源性分别为 97.2%，97.3%。氨基酸同源性分别为 97.3%，98.0％。软件分析表明，各有 22 处和 21 处碱基发生了突变，其中一处碱基发生了缺失，7 个和 5 个氨基酸发生了转换。 本研究不仅为对TGEV进行深入的分子生物学研究提供了资料，为其基因工程疫苗的研制奠定了基础，并且也为该病毒的鉴定提供了一个便捷有效的方法，对猪传染性胃肠炎的防治有重要的现实意义。更多还原

【Abstract】 Porcine transmissible gastroenteritis virus (TGEV) belongs to the family coronaviridae.The virus causes transmissible gastroenteritis (TGE) which does very much harm to the pigfarms. The normal medicines such as antibiotic and antitoxins have no effect to the disease.Vaccination is the effective means for the prevention of TGE.The study isolated RNA fromenteritis tissue. Two pairs of primes were designed to amplify cDNA sequence of M gene by aTranscription-nested Polymerase chain Reaction (RT-PCR)and a Reverse Transcription-nestedPolymerase chain Reaction (RT-nested PCR) . The products of PCR were sequenced andhomology compared .Then they were analysed deeply . The following was the mainexperiments and results: 1. The study isolated RNA from enteritis tissue by Trizol method. After formaldehydegel denaturation electrophoresis, we can see clearly two light bands(28s and 18srRNA) and avague band(5srRNA).The result indicated that the integrality of the isolation RNA was verygood and hardly was degradated.It may be used in RT-PCR. 2. According to the published cDNA sequence of TGEV M gene, with Prime5.0 twopairs of primes were designed to amplify the products which were 1037 bp and 1328 bprespectively by RT-nest PCR based on common RT-PCR. The result demonstrated that themethod has higher specialist and repeation. It can detect correctly virus and avoid falsepositivism. Compared with tranditional methods, such as electron microscopy andimmunization fluorescence technology and ELISE, it is more convenience andcredibility .The results showed that RT-nest PCR may be one method which is neoteric andreliable to detect the RNA virus especially. 3. The assay amplified the full seqence of M gene which is 1 328 bp nucleotide acids byRT-PCR . The amplified product was extracted ， transformed and cloned into pGEM-T<WP=8>Easy vector. The recombinant plasmid was tested by restricted digestion of EcoRⅠand thesequencing. The sequence analysis indicated that the sequence homology of nucleotide acidswas 97.2%, 97.3% and the sequence homology of amino acid was 97.3%,98.0% which wereanalyzed by CLUSTUL(1.82) when it was compared with corresponding sequencing of thedifferent strains in GeneBank. There were twenty-four nucleotide acids mutation and sevenamino acids mutation. And the further analysis indicated that these mutations of nucleotideacids were no meaning and did not affect the fuction of M protein. The study provided not only the important basis for further research of TGEV molecularproperties and the preparation of vaccine, but also an useful viral strain discrimination method.In brief, it provided vital practical value for molecular study of TGEV , development ofvaccine and prevention of TGE as well.更多还原