Study On Spectral Expansion Of Salmonella Hosts By Recombinant T4 Phage

Salmonella is an important zoonotic pathogen,Salmonella infection is the second most common food-borne disease,which causes diarrhea,nausea,vomiting,fever,typhoid and other symptoms.Salmonella is a serious hazard to human health,and it also causes heavy losses to the aquaculture industry.The purpose of this experiment was to compare the cleavage effect of two E.coli phages against Salmonella,and to obtain recombinant phages by homologous recombination,and to determine the host spectrum of recombinant bacteriophages.The tail fiber protein of phage determines the specific host.In this experiment,we designed degenerate primers to amplify the gene sequence of the coding phage tail fiber protein isolated in laboratory,and based on this,we modified the tail filament protein of phage in batch.A recombinant phage that changes or widens the host spectrum is obtained.1?Modification of T4 phage tail fiber protein and determination of recombinant phage host spectrumIn this experiment,two groups of Salmonella were used as hosts,one group was American strain and one group was laboratory isolate.The host spectrum of E.coli phage WG01 and QL01 were detected respectively.The tail fiber protein gene of WG01 was used as donor to reconstruct phage QL01 with narrower host spectrum.Seven genes of different lengths encoding tail fiber protein in phage WG01 were selected to replace the gene sequences of the same position of phage QL01 by gene recombination technique.The recombinant phage WGce could lytic 15 clinical isolates of Salmonella,and its host spectrum was 6 more than that of wild phage WG01 and 9 more than that of phage QL01.Three of the Salmonella strains could not be lysed by wild phage WG01 and QL01.This study provided a theoretical basis for rapid screening or preparation of specific pathogenic bacteria strains from recombinant phages.2?Amplification of virulent phage tail filber protein gene by degenerate primersThe recombinant phage needs a large number of phage tail fiber protein gene sequence to modify the phage tail fiber protein.The aim of this study was to use a conserved amino acid sequence at the N-terminal and C-terminal of the phage tail filament protein,and the amino acid sequence at the N-terminal of the phage tail fiber protein was more conserved.The 5' and 3' ends of the gene37 gene encoding both phage WG01 and phage QL01 tail fiber have different length homologous sequences,the length of which are 190bp and 540 bp,respectively.Degenerate primers were designed by CODEHOP,and 4 upstream primers and 8 downstream primers were designed by this software.32 pairs of primers were obtained by combination.By using the homologous sequence of phage gene37 and blast comparison results,3 upstream primers and 3 downstream primers were designed by this Oligo7.9 pairs of primers were obtained by combination,and 31 strains of bacteriophages isolated in the laboratory were amplified by PCR.The results showed that 9 gene sequences could be amplified from 32 pairs of primers designed by CODEHOP and 9 pairs of primers by O1igo7 could amplify 5 gene sequences,2 of which were clear and 3 of which were vague.The purpose of this study is to simplify the procedure of obtaining phage tail fiber protein gene,reduce the time cost,and provide the necessary gene sequence for further modification of phage tail fiber protein.