| Newcastle disease virus(NDV)is a member of the family Paramyxoviridae and subfamily Paramyxovirinae,can cause fatal and highly contagious Newcastle disease.Naturally infected birds can cause damage to the respiratory system,the reproductive system,and the nervous system.At present,the animals susceptible to Newcastle disease virus have been diversified,no longer confined to poultry animals,and minks also have been found to be infected.Newcastle disease virus has caused great economic loss to mink breeding industry,which has become one of the important diseases that endanger the mink breeding industry in China.Although many scholars have studied poultry Newcastle disease virus,there are still many problems to be solved concerning the biological characteristics and epidemiology of mink Newcastle disease virus.In view of the current method for the detection of Newcastle disease virus are very limited and mainly for the birds,therefore we also need to establish a simple and accurate method for diagnosis,comprehensive and effective monitoring to promote disease prevention and control of the mink industry,which would play an important role in the healthy development of mink breeding industry.In this study,we took the natural incidence mink from the mink farm of Heilongjiang Province as the research object,and used the clinical symptom observation,virus isolation and identification,animal regression test and molecular biological detection method.We verified that the pathogen was mink Newcastle disease virus and its virulence belonged to a strong strain.The mNDV-01-HLJ strain was isolated from mink in China for the first time.Subsequently,the mNDV-01-HLJ genome was successfully cloned and the whole genome sequence of 15192 bp was obtained.The sequence and encoded protein were analyzed,and the molecular phylogenetic tree and bioinformatics analysis were constructed.The results showed that the mNDV-01-HLJ strain belongs to genotype VII of Class II.HN protein is the main capsule membrane glycoprotein of Newcastle disease virus,and the research of HN gene would play an important role in the study of the biological characteristics,diagnosis and prevention of Newcastle disease virus.Therefore,we analyzed HN gene,then target gene was amplified the sequence of 1578 bp in selected good antigenicity area and inserted into expression vector pGEX-6P-1,the recombinant plasmid pGEX-mNDV-HN was transformed into E.coli BL21 induced by IPTG expression.Then soluble expression protein was obtained and its size was about 83 kDa,and could produce specific immune response with rabbit anti mNDV-01-HLJ virus protein antiserum of the laboratory preparation.Finally,we established indirect ELISA detection method of mink Newcastle disease virus using the recombinant protein as a diagnostic antigen,and determined the best condition by matrix method.The concentration of coated antigen was 200 ng/well,the dilution of serum samples was 1:80,the blocking condition was 5%skimmed milk,the working concentration of HRP·labeled rabbit anti-mink IgG was 1:5000,and the substrate action time was 20 min.The experiment confirmed that the established indirect ELISA detection method had good specificity,susceptibility and sensitivity.To sum up,this study is the first time to isolate mNDV-01-HLJ isolated strain from mink and successfully obtain its whole genome sequence,the bioinformatics analysis were constructed.The pGEX-mNDV-HN protein was truncated expression of HN gene of mink Newcastle disease virus,and the indirect ELISA detection method for serum antibodies in mink Newcastle disease was successfully established by this protein.This study will help to analysis the molecular epidemiology of Newcastle disease virus in mink,understand the genetic rule of NDV in mink,and lay a scientific foundation for screening,controlling and diagnosing Newcastle disease virus vaccine strains in mink. |
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