Eukaryotic Expression And Biological Characteristics Analysis Of Mink Enteritis Virus Non-structural Protein Gene NS1

Mink enteritis virus (MEV), which is a member of the family parvoviridae, genus parvovirus, belongs to subgroup of feline panleukopenia virus. The genome of the virus is single-negetive strain. Under natural conditions, MEV was the pathogen of mink viral enteritis which characteristiced by serious diarrhea, principally affecting kits. At present, viral enteritis has also been one of the serious diseases of mink and hampered the development of fur animal farming industry, and specially brought huge economic losses.The NS1 protein of parvovirus plays a key role in virus replication and transcription. Faecal samples confirmed as CPV and MEV infection were collected and used for cloning the complete ORFs genes both of non-structural protein NS1 and the main structural protein VP2 by PCR. The phylogenetic trees of the deduced amino acid sequences of NS1 and VP2 genes were setted and the viruses amino acids variances were summarized, in order to estimate phylogenetic relationships between MEV and others parvovirus with the reference strains which submitted on GenBank. We also forecasted the phosphorylation sites of the NS1 protein. Analysis of amino acids inheritance of the NS1 and VP2 protein of MEV provided further significance to recognize the parvovirus subtypes genetic relationship, the parvovirus genetic variation, cross-species transmission mechanism and control the MEV disease.The whole gene sequence of NS1 protein of MEVB strain was amplificated by PCR. Using the Bac to Bac protein expression system, the recombinant baculovirus which was used for expression of the recombinant NS1 protein was obtained. After identification the success of transfection by PCR, the recombinant NS1 protein (rNS1) expression was identified by indirect immunofluorescence assay, SDS-PAGE, Western blot assay, the rNS1 has satisfactory antigenic specificity.Biological characteristic analysis of rNS1, we found that the isoelectric point and the conformation was similar with the native NS1 protein. The rNS1 was used as coated-antigen to detect the antibody level of NS1 protein in MEV infected mice by indirect ELISA and the serological response to the rNS1 protein was compared with the response of MEV. The result demonstrated that the antibody OD values of NS1 protein rised at 4 day p.i. and dropped at 8 day p.i., lasted until 12 day in MEV infected mice, while the antibody level of MEV peaked at 12 day p.i.. There was almost no antibody detected in inactivited MEV vaccinated mice. Antibody detection of NS1 protein in inactivated vaccine vaccinated minks demonstrated that there was only MEV antibody, but no NS1 protein antibody was detected. Expression and purified NS1 protein provide the underlying basis for establishing distinguish methods between MEV infected and inactivited MEV vaccinated animals, and detecting antibody of NS1 protein in infected animals give scientific prop for controlling MEV.