Cloning And Identification Of S Gene From Transmissible Gastroenteritis Virus Of Swine Strain TH-98

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...