Bioinformatics analysis for gene AS87-01740 in Riemerella anatipestifer and its prokaryotic expression

ObjectiveThis study was conducted to analyze the molecular biological characteristics of gene AS87-01740 in Riemerella anatipestifer and determine the antigenicity of its encoding protein in order to provide new target an-tigens for research and development of R. anatipestifer subunit vaccine.MethodGene AS87-01740 in R. anatipestifer was cloned by PCR,and gene bioinformatics analysis was carried out based on DNASTAR and NCBI database. pCold-TF prokaryotic expression vector was used to induce expression in competent cells of Escherichia coli BL21. Meanwhile,the expression form and antigen specificity of fusion protein were detected by SDS-PAGE and Western blotting respectively.ResultGene AS87-01740 obtained from R. anatipestifer genome amplification contained an open reading frame of 618 bp in total length,encoded 205 amino acids,and its theoretical molecular weight was 21.87 kD,theoretical isoelectric point (pI)8.2,total positively charged amino acids 17,and total negatively charged amino acids 16. Among isolated strains of the same species,AS87-01740 protein sequence had extremely high similarity with serotype 2 isolates(11845,YM,GD and CH-2)(>99%),and 92%similarity with serotype 1 isolates(CH-1 and CH-3). However,in different species,its similarity with Chryseobacterium sp.,Bergeyella zoohelcum and Elizabethkingia meningosepticu was 61%,60%and 57%respectively. The molecular weight of fusion protein obtained by isopropyl β-D-thiogalactoside(IPTG) induction was77 kD ,which was mainly expressed in soluble form. The results of Western blotting revealed that specific immune response could be observed between fusion protein and R. anatipestifer positive serum.ConclusionEncoding protein of gene AS87-01740 is highly conserved in different R. anatipestifer isolated strains ,and can trigger specific immune response to R. anatipestifer positive serum. It is a potential antigen for R. anatipestifer vaccine research and development.