Preparation Of Bovine Pathogenic Escherichia Coli Ghosts And Immunogenic Analysis

Bovine colibacillosis is a common disease in pastures.There are many diseases of dairy cows and calves caused by pathogenic E.coli,such as calf diarrhea,dairy cow colibacillitis mastitis,hysteritis and postpartum sepsis.Escherichia coli is aopportunistic pathogen.E.coli disease is usually caused by feeding management,metabolic diseases,and the environment.Therefore,it is very important to prevent and control of E.coli disease for pastures.Bacterial ghosts?BGs?is a bacterial envelope formed by Gram-negative bacteria losing cell contents.It retains the antigen structure on the cell surface which can stimulate the body to produce an immune response.It is a very good vaccine against bacterial diseases.In this study,the lysis gene E from E.coli bacteriophage phi X174 was used to form a transmembrane pore in the cell membrane.The expression product of Staphylococcus aureus nuclease A?SN?can hydrolyze a large amount of DNA excreted by bacteria into small fragments under 100 bp to prevent the transfer of drug resistance genes and toxin genes in bacteria.In this experiment,a flexible linker with 15bp nucleotides was selected to connect the E gene and SN gene.The target fragment was connected to the vector by homologous recombination.The thermosensitive?p L/p R-c Its857 lysis cassette on the p BV220 plasmid was used to start the lysis system.Centrifuge and wash is carried out after induction of expression.Transmission electron microscopy was used to observe the structure and emptying of bacterial ghosts.Immunological tests on mice were carried out using the prepared bacterial ghosts.There are 6 groups in the experiment.The injection dose of each group is 10⁹ CFU and the injection volume is 100?L.The booster immunization is performed at two-week intervals.Serum was collected after immunization.The indirect ELISA was used to determine the level of E.coli specific antibody Ig G.And 28 days later,20-fold dose of LD₅₀ was used for challenge,and the death of mice was observed in 7 days.The results show that the constructed lysis plasmid p BV220-E-SN can lysis E.coli CE9 ghosts very well,with a lysis rate of 94.8%.The results of transmission electron microscopy shows that the preparation of bacterial ghosts was good,the structure was complete,the interior was empty,and transmembrane pores were visible at end of the bacteria.The culture of the resuspended lyophilized bacterial ghosts showed no bacterial growth.The ampicillin-sensitive E.coli CE9screened was used to determine the median lethal dose(LD₅₀)of Balb/c mice,and the LD₅₀ was calculated to be 2.75×10⁷CFU by the modified Kou method.The results of immunological experiments in mice showed that there are significantly antibody-levels difference between bacterial ghosts subcutaneous injection and formaldehyde inactivated vaccine group,formaldehyde inactivated vaccine+aluminum glue inactivated vaccine group,bacterial ghosts intramuscular injection,oral bacterial ghosts?P<0.01?;the difference between the formaldehyde inactivated vaccine+aluminum glue,inactivated vaccine group and the intramuscular injection group was not significant?P>0.05?.The formaldehyde inactivated vaccine group was significantly different from the oral bacterial ghosts?P<0.05?.The order of antibody levels among the experimental groups is the bacterial ghosts?subcutaneous injection?>bacterial ghosts?muscular injection?>formaldehyde inactivated vaccine+aluminum glue adjuvant>formaldehyde inactivated vaccine group>oral bacterial ghosts.In the challenge experiment,the protection rate of formaldehyde inactivated vaccine,formaldehyde inactivated vaccine+aluminum glue adjuvant,bacterial ghosts?muscular injection?,bacterial ghosts?subcutaneous injection?,bacterial ghosts?oral?and PBS control group are 35%,60%,70%,85%,30%and 0%.The results of autopsy on mice showed that compared with the control mice,the bleeding lung of the mice in each experimental group was significantly reduced,and the edema and bleeding of the duodenum and jejunum are also mild,especially in the bacterial ghosts?subcutaneous injection?group and the bacterial ghosts?muscular injection?group.Consider the antibody level,immune protection rate and necropsy results,the best group is the bacterial ghosts?subcutaneous injection?.In summary,the prevention effect of bacterial ghosts on E.coli disease is better than the traditional formaldehyde inactivated vaccine.Subcutaneous immunization is the best immunization route.