| Salmonella is a common bacterium that can cause zoonosis.Salmonella infection can lead to pullorum,typhoid and other diseases,causing huge economic losses to farms.Antibiotic therapy is an effective treatment for salmonellosis.However,with the extensive use of antibiotics,drug residues and other problems become increasingly prominent.At present,there are many products that can replace antibiotics,and bacteriophage has attracted much attention for its characteristics of strong specificity,fast action,safety and no residue.Phage is a kind of virus composed of protein and nucleic acid.When phage enters the body,it may trigger the immune response,stimulate the immune cells to produce specific antibodies,and affect the function of phage.In this study,lab-preserved salmonella phage GRNsp1 was used to observe the neutralization effect of serum on phage after oral administration and injection,and the changes of specific antibody levels in chicks after oral administration of phage.According to the experimental results,it can better guide the rational use of phage preparation in the breeding process and ensure the use effect of phage preparation.The specific research results are as follows:1.Prokaryotic expression of head deformation protein HMP and tail spur protein P22The whole genome of Salmonella phage GRNsp1 was sequenced and the head deformation protein HMP and tail spike protein P22 were selected for expression after comparison.Recombinant plasmids p ET-28a-P22 and p ET-28a-HMP were constructed.The recombinant plasmids were transformed into expressive receptive state and induced expression.The optimal induction condition was determined as 0.8 mm IPTG induction at 20℃for 8 h,and then purified by Ni-NTA affinity chromatography column.2.Preparation of HMP,P22 protein and phage polyclonal antibodyPhage GRNsp1 was cultured and purified to obtain phage solution with a titer of more than 1010 pfu/m L.The purified HMP protein,P22 protein and phage GRNsp1were immunized to obtain polyclonal antibodies.ELISA results showed that HMP protein,P22 protein and phage GRNsp1 had immunogenicity and could induce humoral immune response of animals.Western Blot showed that both HMP protein and P22protein had affinity for phage GRNsp1 specific antibody,but HMP protein had stronger affinity for antibody.3.Establishment of indirect ELISA assayAn indirect ELISA method was developed to detect phage GRNsp1 specific antibody of using HMP protein as the bottom antigen.The optimal coating concentration of antigen was 0.25μg/m L,the optimal dilution ratio of serum was 1:400,the optimum sealing conditions were 5%skimmed milk for 3 hours,and the optimum action time of serum was 75 min,the optimal dilution ratio of enzyme labeled secondary antibody was 1:5000,and the optimal action time of enzyme labeled secondary antibody was 60 min.4.Animal experimentThe neutralization of the specific antibodies in the serum to the phage was detected.The results showed that compared with the control group,P22 protein,HMP protein and anti-serum of phage GRNsp1 had extremely significant neutralization effect on phage（p<0.01）,indicating that P22 protein and HMP protein played an important role in the phage reproduction process,and the antibody in serum after injection of phage had strong neutralization effect on phage.Inhibits the phage’s ability to infect bacteria.In animal experiments,chickens were fed with salmonella phage GRNsp1 on days1-5 and 26-30,respectively,and serum was collected on designated days to detect the changes of phage-specific antibody levels.Using established indirect ELISA,phage antibody levels began to rise on day 17 and began to decline after peaking on day 23.After the second feeding,the phage antibody increased again on day 35 until day 41,and then decreased gradually.These results suggest that phage GRNsp1 induces a weak humoral immune response in chicks.Neutralization test was performed on the serum of oral phage GRNsp1 group,and the results showed that the serum of OS group had a weak neutralization effect on phage,with a neutralization rate of 0-2%,which was not significantly different from that of the control group（P>0.05）.Thus,compared with injection,oral phage GRNsp1 induces a weaker humoral immune response,and neutralizes phages accordingly,which does not affect the role of phages in infecting bacteria.In clinical use of phage preparation for treatment,oral method can be given priority to reduce phage specific antibody inhibition on phage. |
