Preparation And Efficacy Of The Ghost Of Avian Pasteurella Multocida

Fowl Cholera is a hemorrhagic sepsis caused by Avian Pasteurella multocida which is characterized by acute avian cholera.Clinical symptoms may not be obvious in the early stage,until the late stage of infection,showing depression,feather fluffiness,fever,anorexia,mucus secretions in the mouth,diarrhea and shortness of breath,which greatly endangers the development of the poultry industry.At present,the main methods for preventing FowlCholera are antibiotic treatment and inactivated vaccine and subunit vaccine and attenuated vaccine.The abuse of antibiotics has led to the emergence of a large number of drug-resistant bacteria,and the existing vaccine immune effect is not satisfactory.Therefore,the development of a highly immunogenic vaccine is of great significance to the poultry industry.Bacterial Ghost is an empty shell of bacteria that retains the complete morphological structure and antigen-determining cluster on the surface of the bacteria,but does not contain nucleic acid or other substances,and has good immunogenicity and adjuvant function.In this experiment,the lysis protein E of phage and the nuclease A gene SN of Staphylococcus Aureus were amplified by PCR,which were ligated in 15 flexible amino acids by fusion PCR to form E-15aa-SN（ES）.The ES gene was then cloned into a temperature controlled vector PBV220.The temperature controlled plasmids,PBV-E and PBV-ES,were obtained.The plasmid was used as a template,and the temperature controlled double gene cracking box was amplified by PCR and cloned into Escherichia coli shuttle plasmid to obtain the temperature controlled gene cracking box shuttle plasmids,pBA1100-DLS-E and pBA1100-DLS-ES.The shuttle plasmid was transformed into the prepared competent of Pasteurella multocida by electroporation to obtain a recombinant strain.The recombinant bacteria were cultured at a low temperature of 28°C,and the expression was collected at 42°C.By measuring the growth curve and viable count of the recombinant bacteria,processing cell lysis rate was up to 99.9998%,indicating the successful construction of the BacterialGhost.The LD50 of the Pasteurella multocida C_48-1 was determined to be 0.523 by the modified Karber’smethod.By groping the lysis conditions,double lytic recombinant bacteria and single lysis recombinant bacteria were collected after 2 hoursor 3hoursof inducting.60-day-old Jianghan chicken,which was immunized twice with formaldehyde inactivated vaccine and single-lysed bacteria and double-lysed bacteria vaccine are injected with a 10-fold LD50 dose of Pasteurella multocida C48-1 while the control group was immunized an equal dose of PBS solution.The results showed that all the control groups died after the challenge,the protection rate of formaldehyde inactivated vaccine was 70%,and the protection rate of single-lysis bacteria was 80%,and the protection rate of double-lysis bacteria was 90%,indicating that the Ghost vaccine has a certain protective effect on Jianghan chicken and the immune effect is better than formaldehyde inactivated vaccine.