| Porcine transmissible gastroenteritis (TGE) is a highly contagious, enteric disease of swine caused by porcine transmissible gastroenteritis virus. The main clinical symptoms of infected pigs is vomiting , diarrhea and dehydration. TGE have lead to enormous loss to pig breed .In this study, TGEV was prove to be epidemic in Sichuan distraction by isolating a TGEV strain, genechip of detecting TGEV was constructed and functional study of genes was also carried out.1. Isolation and identification of SC-H strainThe diarrheic material was incubated on the swine testis cell monolayer, the virus induced cytopathic effect (CPE) after five generations passed on the cells , the infected cells began to become swelling, glazing, accretion and floation 30 hours later infected by the virus. The TCID50 of the isolated virus was 10-3.92/0.04ml . The virus could be neutralized by the specific antiserum of TGEV and the neutralization index was 242. Enormous typical virus particles could be observed gathering in the cytoplasm under transmission electron microscope with peplos and 7095nm diameter, the mature virus released to extracelluar by exocytosis .The isolated virus was sensitive to chloroform and ethylether but it was resistant to 5-bromine-2-deoxyuridine and trypsin. the isolated TGEV strain from Sichuan was named SC-H, it provide material for diagnosis, prevelance and control of TGEV.2. Genes cloning and analysis of of SC-H strainPrimers were designed to amplified the complementary DNA of S1, S2, S3 S4, sM , M , N, S',ORF7 and POL respectively. All fragments were inserted into pMD18-T vector to construct recombinant plasmid, these recombinant plasmid were named pMD-S1, pMD-S2, pMD-S3, pMD-S4, pMD-SM, pMD-M, pMD-N, pMD-ORF7 , pMD-S', pMD-POL. Restriction enzymes digestion and nucleotide sequencing were done to identified these recombinant plasmids. The length of S1,S2,S3 and S4 was 1260bp,1280bp,1062bp and 1217bp, after sequencing... |
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