The Prokaryotic Expression Of The SU Gene Of Goat Endemic Intranasal Tumor Virus And The Establishment And Application Of RT-PCR Method And Indirect ELISA

ENTV(Enzootic nasal tumor virus)of sheep and goats is a contagious diseases characterized by neoplastic transformation of secretory epithelial cells in the respiratory tract of the mucosal nasal glands.Clinical signs include continuous nasal discharge,respiratory distress and weight loss.Epidemiological date indicates that ENTV prevalence in the sheep areas such as Inner Mongolia,Hunan and Sichuan,etc.In this research,the method of RT-PCR was established by ENTV-SC Strain,then the SU gene was subcloned into the prokaryotic expression vectors,prokaryotic expression and the initial establishment of the ELISA detection methods.1.Establishment and Application of RT-PCR method for the Detection of Enzootic Nasal Tumor VirusBased on the published sequence of enzootic nasal tumor virus(ENTV)in GenBank,a pair of specific primers was designed and a rapid RT-PCR method of ENTV was established.In result,a fragment of 323 bp,which was expected,was amplified by the RT-PCR method and the homology which compared with ENTV-SC strain in NCBI was 98%.The results that amplified the nasal epithelium of healthy goat.the nasal epithelium of healthy cattle?pneumonococcus and mycoplasma was negative;As low as 6 pg RNA of nucleic acid could be detected accurately.This method had good reproducibility and stability;100 pathological materials had been tested by RT-PCR,and the coincidence which compared with Clinical diagnosis was 100%.The results showed that this method may be used for clinical diagnosis and laboratory research of ENTV.2.Clone and expression of SU gene of enzootic nasal tumor virus from goatSU gene of enzootic nasal tumor virus from nasal secretion of goat was amplified by RT-PCR and cloned into the pMD19-T vector.After sequencing,the SU gene from cloned vector was digested by EeoR I and Sal I and sub-cloned into the pET-32a,the recombinant plasmid was transformed into Rossta.The transformed bacteria were induced by IPTG,and selecting the best condition for the expressed production by SDS-PAGE.The results showed that the recombinant proteins had 60.14 ku,0.75 mmol/L IPTG,37 ? and 5 h of induction time were the best conditions.More pure proteins would be produced after Ni2+ column purification.The purified protein could react with the positive serum of the antibody against enzootic nasal tumor virus from rabbit in a Western Blot test,which showed that the expressed protein had strong reactogenicity.3.Establishment of ELISAA method of indirect ELISA was established based on purified ENTV surface protein of the third chapter.The best conditions were:serum sample(1:40)at 37?for 90min;coating antigen at concentration of 12.5 ?g/ml,37?1h,4? overnight;5%defatted milk powder blocking at 37? 2h;HRP labeled Rabbit anti-Pig IgG(1:4000)at 37?for 40min;the substrate TMB for ELISA being incubated for 15min.The threshold of negative and positive serum was 0.421 by distinguish.The ELISA assay was specific,sensitive,repeatable.