Expression And Identification Of M And NP Gene Of Goose-host Paramyxovirus Strain NA-1 In Prokaryote And Eukaryote

Goose paramyxovirus disease with a high morbidity and mortality,is caused by Goose paramyxovirus(GPMV),a member of Paramyxoviridae. And the clinical symptom and athological changes of the disease is characterized by digestive system disease GPMV can infect all varieties and all ages of geese.The infection spectrum of Newcastle disease virus(NDV)is extending endlessly,In1997 geese infected NDV breaked the traditionary theory"waterfowl don't infect NDV or inapparent infection".From 2000 to 2005 the low virulent strain of NDV was also isolated in Muscovy ducks and swine which caused people cannot but to think highly of NDV. The prevalence of NDV has led to a heavy loss, threatn to poultry industry and become one of the main contagious diseases that restrict the deeper development of poultry industry.In view of gooses or fowl-host APMV-I leading to a severe morbility and death of waterfow threaten the healthy development of poultry industry. Study on the bionomics and genovariation laws of gooses-host paramyxovirus is necessary. which can lay a foundation for the transmission of NDV between different strains.Goose-host paramyxovirus(GPMV)strain NA-1 is multiplied by SPF embryonated eggs to collect allantoic fluid, obtain the purified virus, and to extract the RNA genome of virus. Based on the complete genome sequence of goose paramyxovirus strain ZJ1 logged in GenBank.Two pairs of primers were designed and synthesized,and the complete M gene and NP gene Amplified by RT-PCR.Then the PCR production was cloned and transferred into pGEM-T-Vector. ( named the recombinant"pGEM-M and pGEM-NP"), sequencing and so on were used to identify the recombinant masculine cloning plasmid genetic variation.The results showed that the M gene was 1241 bp long and included a complete open reading frame(ORF)encoding a protein of 364 amino.NP gene was 1746 bp long encoding a protein of 489 amino. Compared M gene of NA-1 strain with other published NDV strains,the average homology of the nucleotide is 82.8% to 99.7%. Compared NP gene with other published NDV strains,the average homology of the nucleotide is 81.1% to 99.7%. These numbers showed that M gene and NP gene of GPMV has a high genetic stability,but has some variations compared with NDV.Based on the complete genome sequence of goose paramyxovirus strain NA-1 and then two pairs specificity primers were designed and synthesized. Through RT-PCR analysis shows that M gene consists of 1 095 bp nucleotides and N P gene consists of 1 470 bp nucleotides. After M and NP gene is cloned into pET-28a vector, the recombinant plasmids, pET-M and pET-NP are transformed into the bacteria BL21 (DE3), and then induced by 1.0 mmoL/L IPTG. Samples were collected at different concentration. The specificity of the expressed protein was identified by SDS-PAGE and Western-blot .The result shows: The optimal amount of M gene expressed protein is induced with IPTG at 1 mmol/L concentration for 4 hours. The fusion protein is about 39.7 Ku in size. The optimal amount of NP gene expressed protein is induced with IPTG at 1 mmol/L concentration for 3 hours. The fusion protein is about 53.2 Ku in size. Both of them have very good antigen.Amplified the open reading frame (ORF) of M and NP gene fragment by the method of RT-PCR.Meanwhile,the target gene fragments were reclaimed and purified to insert into eukaryotic expression vector pCI-neo vector to converted into the E.coli DH5α. then the positive clones were identified by enzyme digestion , PCR and sequencing. The recombinant plasmids of pCI-M and pCI-NP were transfected into Vero cells, and the green fluorescence was observed under fluorescent microscope with the method of indirect immunofluorescent test,indicating that recombinant plasmids of pCI-M and pCI-NP could successfully expressed. After to enlarge culture the Vero cells,the cells were collected and breakdown by the solution of cell disruption, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that specific protein bands about 39.7ku and 53.2 Ku appeared on the gel, Western blotting was performed for identification purpose, the result showed that the bands on the gel were specifically combined with the polyclonal antibodies of Goose-host paramyxovirus strain NA-1,and approved that the expressed product were M and NP protein.