Expression And Enzymatic Characterization Of Riemerella Anatipestifer Nictinamidase

With the development of large-scale duck industry,China has become the largest waterfowl breeding country in the world.Bacterial infectious diseases have become the bottlenecks for the development of duck industry in China.Among them,Riemerella anatipestifer has emerged as one of the major bacterial infectious diseases which hazard the waterfowl breeding in China.R.anatipestifer（also known as duck infectious serositis）is an acute or chronic contagious disease which infects multiple species of birds,including ducks and geese.A total of 21 serotypes of R.anatipestifer have been reported,and the lower cross-protection among the serotypes leads the application of vaccine in a great restriction.So far,the molecular pathogenic mechanism of R.anatipestifer is poorly understood.Nicotinamidases were found in multiple species of bacteria,yeast,protozoa,and plants and are present in many metazoans such as Drosophila melanogaster and Caenorhabditis elegans.Purser,J.E had confirmed that nicotinamidase was an essential enzyme for Borrelia burgdorferi infection,causesd Lyme disease.In Brucella abortus,which causes abortion in domestic animals,the B.abortus nicotinamidase is essential for bacterial replication.Erythrocytes infected with Plasmodium falciparum,a parasite that causes malaria in humans,displayed increased nicotinamidase activity and NAD+ synthesis.Despite the importance of nicotinamidases in diverse biological processes,their precise mechanism of catalysis has not yet to be fully elucidated.Understanding the biological and enzymatic properties of nicotinamidases would aid the control of R.anatipestifer.The paper included four parts:1.Bioinformatics analysis of NAMaseBioinformatics researches showed that NAMase has 201 amino acids with the molecular formula of C1014H1560N262O307S9.It is stable in the solution and the hydrophile of overall protein is rather high.The recombinant protein is a non-secretory protein without the signal peptide.Sub-cellular analysis shows the protein is located in the cytoplasm without a transmembrane region.Epitope analysis reveals that the epitopes of anti-B cells are located at 13-15,20-27,50-67,86,107-121 and 177-187 of the protein,which may be the linear epitopes of B cells.Secondary structure of the protein includes 5 spirals,7 folds and 12 random coils.2.Cloning and expression of NAMase encoding gene from RA strain Yb2Genomic DNA of R.anatipestifer strain Yb2 was used as the template for PCR amplification of NAMase encoding gene at 606 bp,which was then cloned into the expression vector pET28a to construct recombinant plasmid pET28a-NAMase.The recombinant plasmid pET28a-NAMase was transformed into E.coli BL21（DE3）for expression of the fusion protein induced by IPTG.The recombinant protein His-NAMase was successfully expressed at 22.1 KDa in size in a soluble condition.The recombinant protein His-NAMase was purified via Ni+ affinity chromatography for further enzymatic and immunologic studies.3.Preparation of the polyclonal antibodiesRabbits were immunized with purified recombinant NAMase protein to prepare polyclonal antibodies.The antibody titer was tested by indirect ELISA method and the immunogenicity was tested by Western-blot.The results showed that purified NAMase may stimulate specific antibodies in rabbit and the antibody titer could reach up to 1:32000.By Western blot test NAMase protein localized in the cytoplasm.4.Enzymatic characterization of NAMaseEnzymatic characterization of NAMase showed that the optimum pH value is 6;the optimum temperature is 40 ℃;metal ions Zn²⁺,Cu²⁺,Fe³⁺ has an impact on enzyme activity.