The Construction Of Avian Escherichia Coli ETT2 Related Gene Deletion Strain And The Expression And Identification Of Related Proteins

The type ? secretion system(TTSS)is present in many plant and animal gram-negative pathogens,causing damage or allergic reactions to the host or non-host bacteria.The results of the survey showed that the presence of type ? secretion system 2(TTSS)and different pathogenic E.coli may be mutated into the host bacteria.Avian pathogenic Escherichia coli(APEC)can cause pathogenic E.coli disease and bring economic losses to the poultry industry that cannot be underestimated.In order to study the related functions and antigenicity of some gene sites of E.coli type 3 secretion system 2(ETT2),the yqeH,yqeJ,and yqeK genes were knocked out and passed the immune serum.Analysis was conducted to provide reference for further study on pathogenicity of ETT2.The three coding genes of ETT2,yqeH,yqeJ,and yqeK,have certain homology with the related genes of Salmonella SPI-3,which are located in ECs3703,ECs3705,and ECs3706,respectively.To construct the deleted strains of the yqeH,yqeJ,and yqeK genes of ETT2 of avian Escherichia coli 3 strains,specific knockout primers were designed based on the published ECs3703,ECs3705,and ECs3706 gene sequences,using the Red homologous recombination technique for avian E.coli A130512,respectively.The strains were subjected to gene replacement in ECs3703,ECs3705,and ECs3706.The recombinant strain obtained by using FLPe plasmid could be expressed at 30 ? and lost when the temperature was increased,and the aminobenzyl resistance gene carried by FRT homology arm and the chloramphenicol resistance gene carried by FLPe plasmid were eliminated.Finally,the deletion strains that did not contain the resistance gene were screened by PCR and sequence analysis and named as A130512?ECs3703,A130512?ECs3705,and A130512?ECs3706.To further study the characteristics of the proteins encoded by yqeH,yqeJ and yqeK genes,three pairs of primers were designed;the sequences of the yqeH,yqeJ,and yqeK gene epitope enrichment regions were amplified by PCR,and cloned into the pET-30a expression vector.in.Through PCR and enzyme digestion analysis,recombinant plasmids containing the corresponding genes were obtained,respectively.The obtained recombinant plasmid was transformed into BL21(DE3)and subjected to IPTG-induced expression.After SDS-PAGE and Western-blot analysis,the size of the three fusion proteins was 29 kD,19 kD,and 21 kD,respectively.The three fusion proteins obtained were named His-YqeH,His-YqeJ,and His-YqeK,respectively.In order to detect the antigenicity of the His-YqeH,His-YqeJ,and His-YqeK fusion proteins,the expression product was subjected to SDS-PAGE,and the protein containing the target band was excised to immunize healthy rabbits.Serum was collected and separated 7 days after the third immunization;prepared serum was used as primary antibody;horseradish peroxidase-conjugated goat anti-rabbit IgG as a secondary antibody and His-YqeH,His-YqeJ,His-YqeK protein antigens,Western-blot analysis showed that multiple antiserum could react with the recombinant protein.DyLight 800 labeled goat anti-rabbit IgG as a secondary antibody by Western-blot analysis of lysed supernatants and pellets with the original bacterial A130512 and 3 deletions.The results showed that three His-YqeH,His-YqeJ,and His-YqeK polyclonal antiserum did not react with the bacterial lysate supernatant of the A130512 strain and the three bacterial strains A130512ECs3703,A130512ECs3705,and A 130512ECs3706.Reacts with A130512 whole lysate;His-YqeH multi-antibody serum reacts with bacterial lysis and precipitation of A130512 strain and bacterial lysis and precipitation of A130512?LCs3705,A130512?ECs3706,and did not react with bacterial lysis and sedimentation of A130512?ECs3703;His-YqeJ The antiserum reacted with the bacterial lysate of the A130512 strain and the bacterial lysate of A130512?ECs3703 and A130512?ECs3706.It did not react with the bacterial lysate of A130512?ECs3705;the bacteria of His-YqeK and A130512 strain precipitated and A130512?ECs3703,A130512?ECs3705 bacterial lysate reacts and did not react with the bacterial lysate of A130512?ECs3706.