Isolation And Identification Of Muscovy Duck Parvovirus And Cloning And Prokaryotic Expression Of VP Gene

The Muscovy Duck Parvovirus disease is an acute and contagious disease caused by the Muscovy Duck Parvovirus(MDPV),which is characterized by high mortality in 1-3 weeks old Muscovy ducklings.The clinical symptom is diarrhea,dyspnea,exudative enteritis,and the mortality is about 50%to 80%.In 1980,this disease was first reported in Fujian province of China,subsequently, reported in France,America,Japan and in south of China.It has seriously restricted the development of Muscovy duck industry.Epitope,also named antigenic determinant,is the site recognited by antibody in antigen.,which is generally composed of a few directly or in conformation neighboring amino acids,determineds antibody specificity generated by organism,and is base unit to induce humoral immunity.With regard to MDPV,searching protective epitope of virus is substantial for diagnosis,immunization prevention and cure of the disease.VP gene of MDPV overlap with each other and code 3 protein, which are VP1(89KD),VP2(78KD)and VP3(61KD).Their initiation codon locates in 2450nt, 2855nt,and 3044nt respectively.Their terminator codon sites are the same and locate in 4646nt.For the gene length,VP1＞VP2＞VP3.The 3 proteins are capsid protein exposed on the surface of virus. VP2 may be the important protective antigen,whereas VP1 and VP3 constitute important structure of virion and hace poor immunogenicity.In this research,we collected sick poult duck suspected to be infected with Parvovirus,and detected MDPV using PCR products sequencing method.DNA extraction of 20 samples collected in Sichuan,Chongqing,Fujian,Guangxi and Shangdong province was performed,and then PCR amplification was carried out with the primer AL18R2/AL18F2 reported by Chang-kwang Limn. The result showed that 8 samples were positive,which are GX,CQ-2,CQ-16,FL-1,FL-2,DY,FJ and SC-14.PCR products were purified and sequencing,then acquired sequences was compared with MDPV type strain(FM strain)and GPV type strain(B strain).The result showed that only FJ sample had the highest homology with MDPV FM strain,other 7 positive samples possesed higher homology with GPV B strain.Viruse was isolated successfully from FJ sample by inoculating tissue fluid into duck embryos and named FJ strain. VP1 and VP2 gene of FJ strain were amplified with the primer GR/GF reported by CHU CY and MF1/MR1 designed in accordance with MDPV FM strain respectively.Complete VP gene sequence was gained by cloning,sequencing,assembling and correcting,in other words,VP1,VP2 and VP3 sequences are obtained.Nucleotide sequence and deduced amino acid sequence of VP gene were aligned and compared with MDPV FM strain(GenBank accession number:NC₀06147), Guangdong strain(GenBank accession number:AY510603),France strain(GenBank accession number:Z68272)and GPV B strain(GenBank accession number:U25749),Taiwan strain (GenBank accession number:AY382889),then phylogenetic tree was constructed and analysed.The result showed FJ strain shared high homology with 3 MDPV strains,especially with Guangdong strain,99.2%similarity in nucleotide sequence,and 98.5%at protein level.Comparing to MDPV FM strain,the second base of initiation codon in VP2 gene of FJ strain changed T to C,which is identical with initiation codon of GPV VP2 gene(ACG).Hydrophilicity of amino acid deduced fom VP gene was predicted according to Kyte-Doolittle's amino acid hydrophilicity standard applying protean program of DNAStar software.Surface accessibility was predicted by Emini program.Antigen index was predicted by Jameson-Wolf program.Finally,probable epitope was predicted to located in 146-190,240-340 amino acid residue region of VP2 amino terminal on the base of higher structure of MDPV.Primers were designed to amplify the probable epitope region.Products were ligated to pET28b(+)vector,recons were named pFJ.Then prokaryotic expression was induced for recons,and expression protein was obtained and evaluated size by SDS-PAGE electrophoresis,which was 23 KD in molecular weight approximate to theoretical value.