Study On Prokaryotic Expression Of Serotype A Avibacterium Paragallinarum Hmtp Protein And Its Immunogenicity To Chicken

Infectious Coryza(IC)is an infectious disease of the avian respiratory system caused by Avibacterium paragallinarum(Apg).Apg mainly leads to a decrease in the laying rate of diseased chickens and an increase in the elimination rate,which brings huge economic losses to poultry breeding,especially layer breeding.There are 3 serotypes of Avibacterium paragallinarum,amon which the serotype A Avibacterium paragallinarum is the most widely and long-last spread in China.Inactivated vaccines are the most important way to prevent infectious coryza,but large amounts of vaccination will produce side effects that affect the laying and growth of immunized chickens.Studies have shown that Avibacterium paragallinarum has a variety of virulence factors,and hemagglutinin protein is considered the most potential subunit vaccine antigen.In this experiment,the Hmtp protein of serotype A Apg was expressed in prokaryote,purified and prepared into a vaccine,and the immune effect of recombinant protein was evaluated by immunizing SPF chickens,which provided a basis for the prevention and control of Avibacterium paragallinarum subunits.The specific experimental results are as follows:1.In this experiment,the Hmtp gene sequence of Avian Paragallinarum serotype A in Gen Bank was collected,and the sequence alignment was performed by Mega and the gene evolution tree was constructed.The Hmtp gene of the F-8 strain was selected as the recombinant expression gene,the original gene sequence was codon-optimized,and the company was commissioned to synthesize it.The pET28-Hmtp recombinant vector was constructed by homologous recombination and transformed into E.coil BL21(DE3)..2.Through preliminary induction and expression screening,p ET28-Hmtp was finally selected as the final expression plasmid.The induction conditions were further optimized,and it was determined that the best induction program for the soluble expression of recombinant protein was an induction temperature of 30℃,an induction time of 6 h,and an IPTG concentration of 0.5 mmol/L.The recombinant Hmtp protein was purified by a nickel column,The Western blot detection of the anti-A serotype Avian Avian positive serum showed that the recombinant protein can specifically bind to the A serotype Apg positive serum,and it has immunogenicity.3.Remove the salt ions and endotoxins in the purified protein solution.After ultrafiltration and concentration,the final recombinant protein concentration is 1.41 mg/mL.After immunizing SPF chickens,the indirect ELISA test showed that the recombinant protein can induce the chickens to produce specific antibodies,and the antibody level is equivalent to the level of existing commercial vaccines.In summary,in this study,the Hmtp gene sequence of F-8 strain of Avian Paragallinarum serotype A was used as the recombination target gene,and the soluble recombinant Hmtp protein was successfully expressed through the prokaryotic expression system.Both Western blot and animal experiments show that the recombinant Hmtp protein has certain immunogenicity to chickens,which could lay the foundation for further study of the structure and function of Hmtp protein.