Prepartion Of Monoclonal Antibodies Against Duck Mx Protein And Analysis Of Expression Pattern Of Mx In DHAV-3 Infected Duck Embryo

Mx protein was induced by interferon which have the antiviral activity,but the detail antiviral mechanism of duck Mx has not yet been determined.Prepare monoclonal antibodies against duck Mx protein will not only be used to detect it,but also will to provide the necessary material for further research on the structure and antiviral function of Mx protein in waterfowl.The homology of amino acid sequences among different species and the antigenicity of duck Mx protein were analyzed.The genes of Mx-A(1-300 bp),Mx-B(1798-2163 bp),Mx-C(418-939 bp),Mx-CDS were amplified by PCR.The three truncated genes,Mx-A,Mx-B and Mx-C were subcloned into the prokaryotic expression vector pET-30a(+)and pET-32a(+),the recombinat prokaryotic protein rDupET-30a-Mx-A,rDupET-30a-Mx-B,rDupET-30a-Mx-C,rDupET-32a-Mx-A,rDupET-32a-Mx-B,rDupET-32a-Mx-C were expressed after IPTG inducing.At the same time,the eukaryotic expression plasmid pcDNA3.1-Mx-CDS was construted.rDupET-30a-Mx-A,rDupET-30a-Mx-B,rDupET-30a-Mx-C as immunogens to prepare monocleonal antibody,and rDupET-32a-Mx-A,rDupET-32a-Mx-B,and rDupET-32a-Mx-C as detected antigen.Nine hybridoma cells secreting against duck Mx monoclonal antibodies were prepared,they are 2B6,4B6,2D10,3G5,5D5,1C4,3C2,3H4 and 4F7.All of the monoclonal antibodies could react with the detected antigen specifically.Six of them could react specifically with DF-1 cell expressed duck Mx protein by transfected with pcDNA3.1-Mx-CDS,which were 2B6 and 4B6 against Mx-A protein,2D10,3G5 and 5D5 against Mx-B protein,and against Mx-C protein respectively.The results of the subtype identification of monoclonal antibodies showed that the 2B6,4B6,3G5,5D5 and 3H4 strains are IgG1 subclasses,1C4 strain are IgG2 a subclass,2D10,3C2,and 4F7 strains are IgG2 b subclasses.Finally,three hybridoma cells(4B6,3G5,1C4)were selected to prepare ascites and the titers were measured.4B6(against Mx-A protein)monoclonal antibody was used to detect the expression of Mx protein in 6 different tissues of 15 days old SPF duck embryo infected DHAV-3 virus,they were heart,liver,brain,meat,muscle,stomach,intestine.The result revealed the expression level of Mx protein were different in the different tissues which changed with the time.