Identification And Functional Study Of Salmonella Bacteriophage YSP2 Tail Fiber Protein

Salmonella pullorum,as a common poultry pathogen,can cause some acute systemic diseases such as joint inflammation,diarrhea,and loss of appetite in chickens,as well as the most typical symptoms of bacterial white diarrhea.It can be transmitted vertically through eggs,or horizontally through the digestive tract and respiratory tract through feces and drinking water.Infection of Salmonella pullorum leads to the decline of chicken production performance and the hatching rate of breeding eggs.It often causes large-scale morbidity and death of chickens,seriously affects the health of livestock and poultry,and causes severe economic losses.Salmonella is one of the 14 species of animal disease pathogens that the Ministry of Agriculture requires to monitor,and must be eradicated in all breeding farms.In terms of food safety,no salmonella can be detected in all current food standards,which also makes the eradication of Salmonella not only related to the livestock and poultry breeding industry,but also related to food safety and public health safety.In recent years,due to the abuse of antibiotics,Salmonella pullorum has developed resistance to a variety of antibiotics,and has become an important pathogen that affects poultry farming and public health safety,seriously restricting the development of the poultry industry and affecting public health safety.Bacteriophage,as a type of virus that can specifically recognize lysing pathogenic microorganisms,can use their host specificity to specifically detect specific microorganisms.Tail fiber protein is the material basis for phage-specific recognition of the host.Screening and expressing the phage tail fiber protein is of great significance in the detection and eradication of specific microorganisms.In this experiment,after purifying and concentrating the bacteriophage YSP2,a full protein profile analysis was performed using protein peptide molecular identification technology,and as a result,52 protein amino acid sequences were obtained,of which the functions of 15 amino acid sequences were determined.Including tail silk protein,lyase,capsid protein and other common phage proteins.With reference to the protein amino acid database,through homology comparison analysis,it was determined that 5 functional protein sequences were putative tail silk protein amino acid fragments.The amino acid sequence homology of the five-segment tail silk protein is very high,all from Salmonella bacteriophage.The recombinant plasmid was constructed by ligating the target fragment with the vector.Through PCR identification,double enzyme verification,sequence analysis and kanamycin resistance screening,a positive strain capable of expressing tail fiber protein 35 Q was obtained.Through the prokaryotic expression system of Escherichia coli,parameters such as temperature,time and rotation speed were optimized,and the tail fiber protein 35 Q was obtained.The adsorption activity of the tail fiber protein 35 Q was verified by competitive adsorption experiments with phage YSP2.Extract the OMP and LPS of Salmonella pullorum,and verify the targeted adsorption site of the tail fiber protein 35 Q on the bacterial surface by ELISA experiment.The experimental results show that 35 Q has strong adsorption to the OMP of Salmonella pullorum,which proves that the tail fiber protein 35 Q can bind to the outer membrane protein;by destroying the OMP and LPS on the surface of Salmonella pullorum,and then performing adsorption experiments with the phage YSP2.The results showed that compared with Salmonella treated with PBS,the adsorption of YSP2 and Salmonella that destroyed OMP decreased significantly,while the adsorption of YSP2 and Salmonella that destroyed LPS did not decrease significantly.This result also proves that the OMP of Salmonella pullorum is a receptor for adsorption when the bacteriophage YSP2 infects bacteria.In summary,in this study,an Escherichia coli expressing strain capable of efficiently expressing Salmonella bacteriophage tail fiber protein was constructed and obtained.The expressed recombinant tail fiber protein 35 Q has good adsorption activity in vitro.This provides a reference for the targeted detection of Salmonella,and also has a guiding significance for the development of targeted drugs.By exploring the phage receptors on the surface of bacteria and determining the site of action of phage tail fiber protein,it provides a material and structural basis for further understanding of the interaction mechanism between salmonella phage and host.