【作者】 任晓峰；

【导师】 李一经；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2001， 硕士

【摘要】 猪传染性胃肠炎病毒（Transmissible gastroenteritis virus of swine，TGEV）属冠状病毒科冠状病毒属成员，主要引起猪的传染性胃肠炎（Transmissiblegastroenteritis，TGE）。该病是猪的一种急性、高度接触性传染病，主要临床特征是呕吐、严重腹泻和脱水，各种年龄及品种的猪对本病均易感。其中，二周龄以下的仔猪致死率可达100％。预防该病的主要方法是疫苗接种。而传统疫苗在一定程度上存在着排毒散毒、毒力返祖、免疫效果差、需多次注射等固有缺陷，故研制新型基因工程疫苗预防TGE是当前亟待解决的问题。而TGE保护性抗原基因（Spike gene，S gene）的获得则是研制此类疫苗的前提基础。 本试验用猪睾丸（Swine testicle，ST）细胞繁殖并扩增TGEV自行分离株TH-98，根据Genebank中已发表的TGEV S基因cDNA序列，利用Oligo 4.1程序软件设计并合成两对引物，进行逆转录聚合酶链式反应（RT-PCR），分别扩增出2.1Kb和2.3Kb两个片段，经酶切鉴定后，先后插入到pUC18质粒载体上的EcoRI和PstI多克隆位点上，构建了重组质粒pUC-S。根据TGEV S基因位点和pUC18的物理图谱，用相应的限制性内切酶进行酶切鉴定分析，证明克隆的重组质粒为TGEV S基因。对克隆的pUC-S基因进行了序列测定，并与其它五个同源毒株（Purdue-115、Miller、TO14、FS772和96-1933）进行了比较，其核苷酸同源性分别达99％、97％、98％、97％和94％；氨基酸同源性分别达99％、97％、97％、96％和93％。证明了TGEV S基因的保守性。 本研究为更深入地对TGEV进行分子生物学研究及基因工程疫苗的研制奠定了重要基础。另外，也为该毒株鉴定提供了一个有效的方法。更多还原

【Abstract】 Transmissible gastroenteritis virus of swine (TGEV) belongs to the family coronaviridae. The virus causes transmissible gastroenteritis (TOE) in all kinds and age of pigs. Particularly, its mortality in pigs under 2 week old may reach 100%. TGE is a kind of acute, highly infectious disease. TGE?typically clinical manifestations consist of omitting, seriously diarrhea and loss of water. Vaccination is the effective means for the prevention of TOE. However, both attenuated and inactive vaccines exist inherit deficiencies. Such as recovery of virulence, dissemination of virulence, need for adjuvant, etc. So the exploitation of genetic engineering vaccine is the urgent task currently. Furthermore, the obtainment of S gene of TOE is the vital prerequisite for it. In this assay, TGEV strain TH-98 that isolated by ourselves was propagated and harvested on swine testicle (ST) monolayer cell. Two pairs of primers were designed to amplify S gene by RT-PCR according to the published sequence of TGEV扴 gene cDNA with Oligo version 4.1 software. The products of PCR were named Sa and Sb, of 2.1Kb and 2.3Kb respectively. Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in KpnI and PstI multiple cloning sites of the same pUC18 plasmid. The recombinant pUC-S plasmid was identified and analyzed by corresponding restriction endonuclease and nested PCR on the basis of the genetic sites of S gene and pUCI8 plasmid, which was identified as S gene of TOE V. Recombinant pUC-S was sequenced and analyzed in comparison with the other homologous strains. Gene sequence comparison indicated that TH-98 shared 99%, 97%, 98%, 97% and 94% identities with Purdue-i 15, Miller, TO 14, FS772 and 96- 1933 respectively and their amino acid homology were 99%, 97%, 97%, 96% and 93% correspondingly. Therefore, the results affirmed that S gene of TGEV strain TH-98 was extremely conservative. The study provided not only the important basis for further research of TOEV? molecular properties and the preparation of vaccine, but an useful viral strain discrimination method. Postgraduate: REN Xiaofeng Major: Preventive Veterinary Science Advisor: Prof. LI Yijing更多还原