Cloning, Expression And Antibody Preparation Of Rimerella Anatipestifer Hsp15 Gene

Riemerella anatipestifer is a contagious disease, it has a high morbidity and mortality in duck, goose, and various other birds and it has become the most serious infectious disease of duck industry over the world. At present, Riemerella anatipestifer type 1 (RA) is popular, and this study based on the whole genome sequence of RA. Through bioinformatics analysis, the sequence of RA hspl5 protein gene was obtained, the pET-32 a (+) prokaryotic expression vector was used for expression of the protein, further for the preparation of polyclonal antibody against hsp15 protein. The data of this study would lay a foundation for the further research of the disease.First of all, the complete open reading frame was analyzed through bioinformatic way, and physical and chemical properties, the subcellular localization, cross-membrane region, hydrophobic and antigenicity of the protein were predicted. The results showed that the size of RA hsp15 gene was 435 bp, with a Nc value 43.806, and contains a continuous of two rare codon string. The encoding protein was 144 aa, molecular weight of the protein was 16.77 kDa, theory of isoelectric point pI was 7.15. HSP15 protein had no signal peptide or cross-membrane area. Hydrophobic analysis showed that maximum value of hydrophobic is 1.0, the minimum value is-3.4, and the 45-52aa,69-77aa of the protein has a stronger hydrophobicity. Protein antigen epitope analysis showed that the protein antigen epitope mainly in 18aa,23-25aa,28-29aa,33-42aa,66aa,80-84aa,98-114aa and 119-144aa. Two protein kinase C phosphorylation sites and four casein kinase Ⅱ phosphorylation site were found through functional sites analysis.In order to conduct the thorough research to the RA hsp15 protein, the RA hsp15 gene was amplified through PCR, then cloned into T vector, after identification, the RA hspl5 gene segment was cloned into prokaryotic expression vector pET-32a, and the recombinant prokaryotic expression plasmid pET-32a/HSP15 was then successfully constructed. The plasmid was transfered into E. coli BL21 bacteria. Using IPTG induction and SDS-PAGE analysis, the molecular weight of the recombinant protein was 37 kDa, consistent with expected size. The recombinant protein is soluble. At the same time, the induction temperature, induction time and IPTG concentration has been optimized to determine the best conditions, and 0.4 mmol/L IPTG,37℃ induction for 6 h was the best expression condition. Western-blot test showed that the recombinant protein has good immunogenicity.The recombinant protein was purifiedthrough affinity chromatography purification, and the purified protein has high purity, using the purified protein mixed with the same amount of freund’s complete adjuvant and incomplete freund’s adjuvant to immune rabbit for polyclonal antibody preparation. Agarose diffusion test showed that the titer of the rabbit-anti-HSP 15 serum was 1:8.