Protein Gene Discovery. Riemerella Anatipestifer Capsular Polysaccharide Output And Its Immunogenicity

Riemerella anatipestifer infection is a contagious disease of domestic ducks, geese, turkeys and various other domestic and wild birds. It is a major disease confronting the duck industry throughout the world. It accounts for significant economic losses due to high morbidity and mortality. The serotypes of RA are complex and more than 20 serotypes have been reported and lack cross-reactions with each other. To date, studies on RA are very limited and no report about the gemomic library of RA serotype 1 domestic. So we constructed the gemomic library of RA serotype 1 due to its high frequency of occurrence in China. We expect to gain antigen on basis of screening the constructed gemomic library. It maybe used as valuable candidate of vaccine and specific antigen for diagnosis and epidemiological surveillance.The total genomic DNA of CH-1 strain of Riemerella anatipestifer serotype 1 was partially digested with Sau3A I and the 1.0kb-6.5kb DNA fragments were recovered from agarose gel. The DNA was ligated with ZAP Express Predigested Vector by T4 DNA ligase and packaged by GigapackIII Gold packaging extract. The results showed that the packaging titer was 5.23×10~6 pfu/ml and recombinant rate was 96.8%.It is concluded that the constructed genomic library can be used to screen target clones. The constructed genomic library was screened by using the sera of immunized rabbits. A phagemid was gained with in vivo excision of the positive plague. The phagemid was sequenced and then submitted to GenBank with accession number of DQ151838.The submitted nucleotide sequence was analyzed with bioinformatics method including BLAST and FASTA program, ORF finding, gene prediction. It concluded that the ORF1110 was a complete open reading frame of 369 amino acids. It was a complete gene sequence relative to capsular polysaccharide export protein. The other ORF of 384 amino acids named ORF 1155 was related to outer membrane protein, but the ribosome binding site was not find from its upstream before initial start codon. So it needed more research to decided total sequence length. Further analysis with proteins alignment, subcelluar localization, transmembrane and hydrophobicity prediction showed that ORF1110 was related to capsular polysaccharide biosynthesis closely. The ORF 1155 was related to outer membrane protein and surface antigen family. Antigenicity prediction with...