Sequencing Analysis Of E2 Gene Of Muti-Passsaging Spleen Virus And Cytopathogenic Cell Virus And Proof-Testing DI In Cytopathogenic Cell

This text makes use of RT-PCR and nPCR techniques, mensurate nucleotide sequence E2 gene of CSFV Shimen which muti-passaging in pig and induced endothelial cell pathological. Compared and analyzed it's nucleotide and amino acids sequence homology with standard CSFV Shimen.So we can know E2 gene of CSFV variability and provide molecule epidemiology information for prevention classical swine fever.At the same time,swine umbilicus veins was infcted by CSFV Shimen,after induced pathological changes, checkout DI by Northern blotting,this can provide evidence for study pathogenesis of CSFV.Following is experimentation and results. (1) The E2 gene of CSFV which muti-passaging in pig and induce cell pathological. were amplified by reverse transcription polymerase chain reaction (RT-PCR) and the nested PCR(nPCR),Then them were cloned and sequenced. Compared and analyzed it's nucleotide and amino acids sequence homology with standard CSFV Shimen by DNA star software. The results showed that the nucleotide homology between spleen virus and Shimen was 99.4%,the aminoacid homology was 99.0%. It was showed epitopes of all strains were not variable obviously by analysising the variation of some main amino acid residues substitutions of E2 gene main antigenic domains. (2) Swine umbilicus veins was infected by spleen virus and induced pathological changes. The E2 gene of cell virus were cloned and sequenced. The results showed the nucleotide homology between cultured cell virus and Shimen was 98.3%,the aminoacid homology was 96.7%。It was showed epitopes of all strains were also not variable obviously by analysising the variation of some main amino acid residues substitutions of E2 gene main antigenic domains.But find that the variability of spleen virus little than cell virus,this showed that CSFV genetic of muti-passaging in pig are stable than cultured cell. (3) According to published article, we designded two pairs of primer to magnified p80 gene,use DIG Random Labeling and Detection Kit to label p80 gene as probe. Swine umbilicus veins was infected by CSFV Shimen, ,after induced pathological changes,we distill total RNA in 36 hours,54 hours,72 hours. Electrophoresis of RNA through Agarose Gels Containing Formaldehyde,then transfer it to nylon membrane, hybridize RNA by Northern blotting method. The result showed there was no deleted genome in pathological cell RNA,so we can deduced that there no DI in short-term cytopathogenic cell.