| Avian colibacillosis is caused by avian pathogenic Escherichia coli(APEC),resulting in the death of embryos and chicks,sepsis,air sacculitis,pericarditis,salpingitis,enteritis,peritonitis and coligranuloma,etc.Due to the serotypes of pathogenic E.coli are numerous and its antigenic structure is complex,avian colibacillosis is very difficult to treat.Therefore,seeking for the antigen of main APEC and simulate their natural infection of mucosal immunity,is of great importance.In this study,for screening lactobacilli that can cause mucosal immunity,deliver specific antigen and analyzing their sensitivity to antibiotics,intestinal content were extracted from healthy chicken and plated onto MRS-Ca CO3 medium plate,anaerobically cultured at 42℃,and then the gram positive,non-movement and rod-shaped bacteria were selected and subjected to analysis via catalase, oxidase and nitrate reduction activity tests, and sugar fermentation reaction-based phenotypic identification,and sequence analysis of 16 S r RNA amplified by PCR assay.Results showed that there were 8 Lactobacillus strains obtained from the intestinal tract of chicken,including 3 Lactobacillus crispatus strains,2 Lactobacillus johnsonii strains,2 Lactobacillus salivarius strains and 1 Lactobacillus saerimneri strain.All these strains were able to grow well at 37 ℃ and 42 ℃, and showed certain anti-Gram positive and Gram negative bacteria activity.Adhesion experiments in vitro showed that all of the isolated Lactobacillus strains had adhesion ability and could colonize in intestinal tract.Furthermore,all these strains were sensitive to many kinds of antibiotics.The genes encoding APEC serotype O1 fimA and outer membrane protein C(Omp C) were selected as target.Using the genomic DNA of E.coli as template,the fim A gene of 528 bp was amplified by PCR with primers A1(containing Apal I site and sequences encoding linker) and A2(containing Apa I site).At the same time,the Omp C gene of 1074 bp was amplified by PCR with primers C1(containing Sac I site and sequences encoding Flag label) and C2(containing Apal I site),and then,the fimA gene and Omp C gene were subcloned into p MD18-T vector,giving rise to recombinant plasmid p MD18- T-Omp C-fim A.Then,the fusion gene OmpC-fim A was cloned into the constitutive expression plasmid pPG-T7g10-PPT constructed by our lab,generating the recombinant plasmid pPG-T7g10-PPTOmp C-fimA.Then,the recombinant plasmid was transformed into Lactobacillus casei 393 and Lactobacillus saerimneri(M-11) isolated in this study via electroporation method,respectively,giving rise to the recombinant Lactobacillus strain p PG-T7g10-PPT-Omp C- fim A/L393 and pPG-T7g10-PPT-Omp C-fimA/ M-11.Result showed that the fusion protein of an about 101 k Da was expressed by the two recombinant lactobacilli detected by Western blot,respectively.The colonization characteristics in chicken intestine of the recombinant lactobacilli were analyzed,and results showed that daily weight gain of the chicken received the recombinant lactobacilli was significantly increased compared to the control group; the recombinant lactobacilli could survive in intestinal environment of p H 3.0 and grow well in 0.1% bile salt environment; on days 14 post oral administration,the number of recombinant bacteria remained stable.and it can last to days 28.In summary,this study obtained 8 isolates of lactobacillus from the intestinal tract of chicken.constructed the recombinant lactobacillus constitutively expressing fusion fimA and omp C of chicken pathogenic Escherichia coli.The recombinant bacterias can colonize in the intestinal tract of chicken and have the characteristics of probiotics.The recombinant strains are ideal candidate vaccine to the prevention and control of avian colibacillosios caused by avian pathogenic Escherichia coli. |
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