Sequence Analysis Of F Gene And Establishment Of Nucleic Acid Probe For GPMV

Goose Paramyxo virus disease is regarded as one of the most devaetating diseases of poultry in the word, caused by Goose Paramyxo Virus. It is included as an Office International des Epizooties(OIE)list A disease.In the past two decades,an intensive vaccination program against Goose Paramyxo virus disease had been practiced in both large-poultry operations and village poultry farming.This disease breaks out infrequently in some vaccinated flocks,however. And atypical Newcastle Disease in the chicken flocks have been more and more frequently reported.Even some goose flocks with high antibody level also suffer ND.One Goose Paramyxo Virus(named LS-1) was isolated from Liang shan. The Fusion (F) gene of this isolate strain was amplified by reverse transcription-polymerase chain reaction (RT-PCR) technique.The F gene was sequenced after the products of amplification being cloned.So that it can give great help for the study of the prevalence and control.This study included three parts.PartⅠ: Isolation and identification of GPMV LS-1 strain and studies on it's biological characteristicsOne strain of GPMV was isolated from Liangshan ,this strain was able to agglutinate chicken red blood cells,which can be inhibited and neutralized by New castle disease virus (NDV) classic antiserum, but can't be inhibited by avian influenza virus (AIV) classic antiserum (H9 subtype). It's MDT,ICPI and IVPI are 39.5h, 1.76, 2.42 respectively.PartⅡ:Molecular Cloning and Analysis of the Fusion Protein of GPMV LS-1 Strain Two pairs of primers were designed for amplifying the F protein gene in RT-PCR. The Fusion protein gene was amplified and cloned into pMD18-T vector. The sequence of F protein gene was determined and it's length was 1653 bp, coding 551 amino acid (AA) , having 6 glycosylation sites. The amimo acid sequence in the cleavage site of the deduced peptide was 112Arg-Arg-Gln-Arg-Arg-Phe117,matching to that of all virulent strains. Compared with published GPMV strains , this sequence homologous rates of nucleotide and deduced amino acids were 87.0% to 98.4% and 87.0% to 98.5%.PartⅢ: Establishment and Application of Digoxigenin-Labeled Nucleic Acid Probe for GPMVone pair of primers were designed for amplifying the 856bp fragment in RT-PCR. The PCR product was labeled with digoxigenin as cDNA probe for detecting the GPMV .The result of the detection showed that the RNA of the GPMV were positive , the other virus and bacteria were negative. The sensitivity result showed that as few as 3pg/μL of GPMV could be detected by DIG-labeled probe . The detection results for clinical samples tissue showed that GPMV could be found in spleen,trachea,lung and liver. It was proved useful in diagnosis and provided a good method to epidemic investigation.