| Enzootic nasal tumor virus(ENTV)is the etiological agent of enzootic nasal adenocarcinoma(ENA).ENTV associated with neoplastic transformation of epithelial cells of the ethmoid turbinate.ENTV-1 can infect sheep,and ENTV-2 can infect goats.Because lack of an infectious molecular clone and the inability to culture the virus,the pathogenic mechanism of ENTV has yet to be completely confirmed.The full-length genes of ENTV-2were very few in Genbank.Epidemiological date indicated that ENA were prevalent in Inner Mongolia,Hunan,Sichuan,Chongqing,Shaanxi,etc.In order to give further insight regarding the properties of the ENTV,the main research contents include these aspects:Diagnosed the goats with tumor in some goat farms in Shaanxi province,and establishment of an RT-PCR method for detection of ENTV-2;Cloning and sequence analysis of full-length genes of 4 ENTV-2 isolates from different areas;Preparation of polyclonal antibody against Gag or Su protein,respectively.1.In order to diagnose the goats with tumor in 4 goat farms in Shaanxi province,clinical and pathological observation have been done.The cases were diagnosed with ENA.The RTPCR Method for detecting ENTV-2 was established.The positive rate of clinical samples detection is 15.5%.2.The complete sequences were determined from four isolates of Shaanxi province through the subsection cloning.These sequences were analyzed.The ENTV-2-Shaanxi genomes shared greater than 98.2~98.7% sequence identity with ENTV-2-SC(accession number HM104174.1),and 89.6~89.8% sequence identity with the ENTV-2 European strains sequences(accession number AY197548.1).The main sequence differences between these viruses reside in LTR,two variable regions of gag,Orf-x,and the transmembrane(Tm)region of env.Most of the amino acid differences were found in Orf-x,which in the corresponding ENTV-2-Shaanxi and ENTV-2-SC genomes were 108 amino acids,but ENTV-2 sequences(accession number AY197548.1)are 166 amino acids.Apart from Orf-x,two small regions of Gag,Tm region,especially the cytoplasmic tail(CT)of Env,were the majority of differences between ENTV-2-Shaanxi1~4 and other ENTV-2.A stretch of 6consecutive proline residues exists in VR1 of the ENTV-2-Shaanxi1~4 isolates.All the ENTV-2-Shaanxi isolates have the YXXM motif in the cytoplasmic tail of the Env,andYXXM motif is the binding motifs of phosphatidylinositol 3-kinase(PI3K).Phylogenetic analysis by nucleotide sequences showed that four ENTV-2 isolates of shaanxi province were closest related to two ENTV-2 isolates published in NCBI,especially with ENTV-2-SC strain.3.The gag and su gene was amplified by PCR.The target genes were cloned into pET-28a(+)vector,respectively.The plasmids pET-gag and pET-su were transformed into E.coli Rosetta(DE3),respectively.The expression of recombinant plasmid in Rosetta(DE3)were induced by IPTG and detected,respectively.The expressed protein were purified by Ni-NTA affinity chromatography and refolded,then vaccinated the mouse,polyclonal antibody against the Gag and Su protein were obtained,respectively.Western blot analysis showed that the recombinational fusion protein could react with the polyclonal antibody specifically,respectively.The purified protein can’t react with the serum of ENA. |
