Cloning, Molecular Characteristics Analysis And Prokaryotic Expression Of The P1Gene Of Yersinia Ruckeri

Yersinia ruckeri （Y. ruckeri） is one of the most prominent pathogenic bacteria which is harmful to aquaculture all over the world. As one of the virulence factors of Y. ruckeri, Yrp1protein has contributed importantly to its pathogenicity. However, there is little information about the molecular characteristics of the Yrpl protein or the gene encoding it. In order to get the bioinformatics data and provide the theory basis for the further study of related function and pathogenic mechanism of them, the p1gene of Y. ruckeri （yrp1） was amplified by PCR with specific primers which were designed according to the sequence of the yrp1gene in GenBank and inserted into pMD19-T vector, followed by DNA sequencing. Molecular characteristics analysis of the yrp1gene and the protein which is encoded by it was performed by bioinformatics tools. Then the yrp1gene was subcloned into pET-32a（+） and transformed into BL21（DE3）, followed by induction with IPTG and optimization of induction process conditions, such as IPTG concentration, culture time and temperature. These works might lay the foundation for large-scale preparation of the Yrp1protein vaccine.The result of bioinformatics analysis showed that the yrpl gene was1434bp in length and contained a complete ORF. The Yrpl protein which is encoded by the yrp1gene was made up of477amino acid. The theoretical relative molecular mass and iso-electric point of the Yrpl amino acid sequence were about51.5kDa and4.48seperately. Molecular formula of it was C2284H3422N608O742S8. The protein with higher level of Gly and Ser and lower level of Trp, Met and Cys does not contain Pyl and Sec. This protein has a amino acids composition of230hydrophobic amino acids,247hydrophilic amino acids,55acidic amino acids and28basic amino acids, accounting for48.2%,51.8%,11.5%and5.9%separately. The Yrpl protein which contains a ZnMc superfamily conserved domains is most closely related to Serralysin of Yersinia ruckeri （GenBank accession number ZP₀4616595）. The result of hydrophobicity prediction analysis revealed that the hydrophilic regions of the polypeptide were more than the hydrophobic regions, but recombinant protein solubility prediction indicated that the solubility of the recombinant protein was only35.9%when E.coli was selected for induced expression. The polypeptide contains some important sites related to post-translational modification, including35potential phosphorylation sites and4potential N-glycosylation sites. The Yrp1protein does not contain a signal peptide, even though it is a secretory protein. The result of secondary structure prediction showed random coil,(3-sheet and a-helix are in charge of52.62%,25.58%and17.4%respectively. The three-dimensional conformation of the Yrp1protein is mainly consisted of random coil, β-sheet and a-helix.The analysis of codon bias indicated that the codon usage frequency of the yrpl gene was distinctly different and it preferred to perform in yeast and E. coli.The result of induced expression revealed fusion protein with relative molecular mass of approximately72kDa was mostly packaged into inclusion bodies. The optimization of induction process conditions led us to perform the recombinant protein induction at37℃for4h, with0.8mmol/L IPTG.