Bioinformatics Analysis And Expression Of Riemerella Antipestifer RagB Gene

Riemerella anatipestifer (RA) can cause ducks, geese, turkeys and other poultry duck plague creamer fungus infection. The disease is a kind of is acute or chronic septic after contact with sexually transmitted diseases, and is currently one of the important diseases endangering the duck industry. In this study, what I did is the ragB gene (Genbank accession No.:CP003787.1) from RA CH-1 strain focuses researches on bioinformatics analysis, cloing and expression of RA ragB gene. The main results are as follows:1. Bioinformatic analysis of Riemerella antipestifer ragB geneThe whole ragB gene length is 1593 bp of Riemerella anatipestifer. the G+C content of the gene sequences is 35.72%. The gene contains a open reading frame is ORF1593. The effective number of codons (ENC) is 38.491. This ORF contains 43 rare codons,3 codon two couplet but non continuous more than two rare codons string. Fifty-nine codons (excluding Met, Trp and the termination codons) in the polypeptide, with eight synonymous codons strong bias toward A-ended and twelve toward T-ended at the third codon position, were used. There are 25 codons showing distinct usage differences between RA ragB and E. coli,30 between RA ragB and Homo sapiens,26 codons between RA ragB and yeast. Therefore the yeast and E. coli expression system may be suitable for the expression of RA ragB gene if some codons could be optimized.RA RagB encoding 530 amino acid protein with theoretical molecular weight of 58.97 kDa and isoelectric point pI 5.716, which contained 2 N-glycosylation sites,5 Protein kinase C phosphorylation sites,3 Casein kinase II phosphorylation sites,9 N-cardamom acylation site,2 Tyrosine kinase phosphorylation site,1 cAMP and cGMP dependence site, and 1 signal peptide but without transmembrane domain. Analysis of the antigenic protein indicates that the 60～77,186～201,251～269,387～403,495～513 amino acids has a continuous strong antigenicity.The subcellular localization analysis showed that the protein is mainly distributed in the outer membrane. Phylogenetic analysis showed that amino acids of the protein with other members of Flavobacteriaceae RagB protein have a low similarity.2. Prokaryotic expression RA ragB geneWe used Oligo 7.1 designed a pair of primers and used to amplify the ragB gene which fragment is 1623 bp length. A pMD19-T-ragB cloning vector and a pET-32a-RagB expression vector were constructed, and expressed in E.coli BL21 system. The expression conditions is:37 ℃,0.8 mmol/L IPTG final concentration induced expression 10 hours, under optimal conditions can be a lot of recombinant protein expression. A 80 kDa fusion protein RagB was expressed by SDS-PAGE analysis. and mainly expressed in inclusion body in the form of insoluble. The results of Western blot and SDS-PAGE analysis with rabbit anti RA CH-1 positive serum show that the recombination protein has a better reactogenicity.