The Prokaryotic Expression And The Preparation Of Monoclonol Antibody Of N Protein Of Canine Distemper Virus

Canine distemper(CD)is a highly contagious epidemic diseases caused by canine distemper virus(CDV),spreading through direct or indirect touch.It's featured with double-phase fever,severe intestinal and respiratory tract infection when host infected with CDV.In some cases with severe infection,host might have neurological syndrome or even developed to death.The death rates in CDV infection are various depending on the ages and species of hosts,which in unvaccinated domestic pet dogs could reach 80%.CD now is posing a serious threat in protecting domestic pet dogs or surname and etc.,and also in wild animals like pandas or tigers.However,into the researches in CD in labs,The detect on the infection of CDV mostly based on the observation on cytopathic effect in cellular morphology,which could be subjective and inaccuracy since it's based on the judgment from observer.Meanwhile,the direct immunology fluorescent antibody buying abroad could be time consuming and expensive.This research aims to attain the construct protein N of CDV in vitro.,as an antigen to stimulate mice producing B lymphocyte cells.Afterwards via the process of cellular fusion,we gain a sustainable antibody generating hybridome cells.Monoclonal antibody(m Ab)generating from mice injected in abdomen with this protein.This m Ab could help build a kind of direct fluorescence immunity measures.Research in this paper aims to attain DNA nucleotide sequence of CDV construct protein N from the virus which is conserved in labs with designed primers,using RT-PCR measures.The prokaryotic expression vector p ET-22b-CDVN was resulted from the connection between the prokaryotic expression vector p ET-22b(+)and achieved CDV N DNA nucleotide sequence by enzyme digestion and connection.With the guarantee of correct connection between N and initiation codon of vector and the absent of any mutation in N,using appropriate concentrate of IPTG to stimulate E.coli Rosetta(DE3)to generate the construct protein of N.After gain this protein in amount and a series of steps to purify and renature it,using it as an antigen to inject in mice with appropriate immune procedure and immune measures;Thus,immune mice could produce B lymphocyte generating antibody against this protein.To gain a cell type CDVN-SP2/0 which could sustainable secret antibody in those hybridome cells,filtrating and subcloning could be necessary.As the process artificial secreting tumor need a certain amount of cells,culturing CDVN-SP2/0 in vitro.for enlarging cells,and counting and diluting which to an appropriate concentration.We finally gain m Ab in abdominal dropsy from the secreting tumor in mice resulting from injection of talked CDVN-SP2/0 cells.In this research we gain the gene of CDV construct protein N,and the protein N of CDV.After all,we gain a cell strain sustainable secreting this N fusion protein and the m Ab generating from it,which could have great potential in the promoting CDV research.