Evaluation Of Immune Efficacy Of Salmonella Gullinarum Ghosts

Fowl Typhoid(FT)is a septic infectious disease caused by Salmonella gullinarum(SG).Poultry of all ages are susceptible and the mortality rate can reach 10%-50%.Due to the poor breeding management level in China's aquaculture industry,the spread of Salmonella gullinarum between livestock and poultry cannot be effectively controlled,causing huge economic losses to the aquaculture industry.The more common production is the live attenuated vaccine of Salmonella gullinarum,but the live attenuated vaccine is less safe and may cause pollution to the environment.Therefore,it is very important to develop a safe vaccine with high immune effect.Bacterial ghost refers to dead bacterial cells lacking nucleic acid,cytoplasm and cytoplasmic contents.It is used to liysis bacteria by physiological and biochemical methods.Under pressure,bacterial contents flow out and form empty bacterial shells.Compared with traditional vaccines,Bacteria ghost completely retains the antigen structure on the surface of bacteria,so it has high safety and immune effects.At the same time,it has adjuvant properties,and can load foreign antigens,DNA vaccines,drugs and other substances.And the preparation method is simple and fast,so it is a very good candidate for inactivated vaccine.This experiment intends to clone E gene and SN(Staphylocooccal nuclease A)gene by PCR method to construct two split plasmids.At the same time,the suicide plasmid-mediated method was used to construct the asd gene deletion strain of Salmonella gullinarum,and the expression of the lysed plasmid was induced to prepare bacterial ghosts.Through animal experiments to evaluate the immune effect of bacterial ghosts,it provides a new method for controlling the spread of Salmonella gullinarum and reducing economic losses in the breeding industry.1.Construction of lysed plasmid and asd mutant strains of Salmonella gullinarumObjective:To construct a lytic plasmid with excellent lytic performance and asd gene deletion strains of Salmonella gullinarum.Methods:The E gene was cloned by PCR amplification,then the cleavage plasmid p BAD+E was constructed by enzyme digestion.At the same time,Staphylocooccal nuclease A gene was introduced,and it was fused with E gene by Overlap PCR,and then the cleavage plasmid p BAD+ES was constructed by enzyme digestion and ligation.The suicide plasmid was constructed by using wild strain of Salmonella gullinarum as a template,and the asd deletion strain of Salmonella gullinarum was constructed by suicide plasmid-mediated method.Conclusion:The lytic plasmids p BAD+E and p BAD+ES,as well as the asd mutant strains of Salmonella gullinarum were successfully constructed.2.Preparation of Salmonella gullinarum ghostsObjective:To efficiently prepare Salmonella gullinarum ghosts.Methods:The lysed plasmids p BAD+E and p BAD+ES were electrotransformed into competent cells of Salmonella gullinarum,then positive transformants were selected by antibiotic screening and gene sequencing comparison.The bacteriolysis kinetics test was carried out on the positive transformants to determine the optimal induced lysis conditions.At the same time,the pros and cons of the two lysed plasmids were compared to determine the most efficient preparation method for the Salmonella gullinarum ghosts.Finally,the morphological structure of the ghost was observed by scanning electron microscope.Conclusion:Through experiments,we can find that the performance of double-lysed plasmid p BAD+ES is better than single-lysed plasmid p BAD+E in terms of lysis rate and induction time.When the positive strain was cultured to an OD₆₀₀ value of about 0.4,after 2 hours induction,the lysis rate was the highest,and the lysis rate was up to 99.85%.Scanning electron microscope observation showed that the overall structure of the bacterial ghost was complete,and one or several obvious Bacteriolytic pores.3.Evaluation of the immune effect of Salmonella gullinarum ghostsObjective:Evaluation of the immune effect of Salmonella gullinarum ghost vaccine in the chicken models.Methods:Sixty 4-day-old Roman chicks were randomly divided into three groups:the bacterial ghost vaccine group,the inactivated vaccine group and the blank control group,with 20 in each group.One week after the adaptive breeding,the first immunization was carried out at 10 days of age,and a total of 2 immunizations were given at intervals of 2 weeks.Blood was collected from wing veins 3 days before immunization,10days after the first immunization and 10 days after booster immunization,and the serum was separated.The extracted salmonella gullinarum outer membrane protein OMP was used as the coating antigen,and the serum Ig G antibody level in the immunized chicken was detected by indirect ELISA.Two weeks after the second immunization,challenge was performed with wild-type Salmonella gullinarum.The number of chicken deaths was observed and recorded daily for 14 days after challenge.Based on the chicken survival rate,visceral pathological changes,and humoral immunity levels,the immune protection effect of Salmonella gallinarum ghosts vaccine was comprehensively evaluated.Conclusion:Salmonella gallinarum has no obvious stress response after immunization,which can induce the body to produce a good humoral immunity,and the immune protection rate can reach 67%.