| Avian pasteurellosis, also known as fowl cholera or the poultry hemorrhagicsepticemia, caused by Pasteurella multocida, is a contact, septic, infectious diseases.Itcan be infected bird species such as chickens, ducks, geese, turkeys and quail.Thediseases with a sudden and popular feature are created high morbidity and mortalitycausing huge economic losses in the poultry industry. The diseases with short diseasecycle and high mortality rate are not easy to treat, are listed as one of the focus controlpoultry diseases, prevention and control of the epidemic has become essential.Vaccination as an effective measure to control pasteurellosis, scholars has beenattached great importance to the diseases prevention and control research over theyears. The development of effective vaccines has become a hot topic.In recent years, the study of avian Pasteurella multocida antigens are mainly inthe capsule, fimbriae, lipopolysaccharide, pili and outer membrane protein, however,the force of the protection afforded by each antigen is limited. Efficient vaccine ofavian pasteurellosis should make as much as possible with a variety of features ofrecombinant antigen in the vaccine. In this study, the Expression Library of avianPasteurella multocida genomic library were constructed, by immunization with mice,immune protective antigen gene was screened, which lay the foundation for thedevelopment of new vaccines, and a single fragment with protective antigen gene wasextracted from the library, by prokaryotic expression analysis, a new gene was foundwhich paved the way for further in-depth the vaccine development.In this study, the genome was extracted from strains of avian Pasteurellamultocida CVCC474, digested into500-3000bp DNA fragments using the enzymeSau3AI, inserted into the the restriction site of BamH Ⅰ, transformed into Escherichiacoli DH5a host. The DNA expression library total capacities were105clones and waodivided into Five DNA expression librarys. Recombinant plasmids were extracted respectively from the five DNA expression libraries, and then purified and dissolved inPBS.1056-week-old inbred female mice were randomly assigned to7groups and15mice in each group. Group1-5were the experimental groups and the empty vectorpcDNA3.1(+) group and PBS were control groups. Groups1-5were inoculated therecombinant plasmids extracted from sub-library1-5respectively. The recombinantplasmids diluted with sterile1×PBS (pH7.2) and the concentration was100μg/μL.The group of pcDNA3.1(+) inoculated with the plasmid pCDNA3.1(+) which wasdiluted by sterile1×PBS (pH7.2) with the concentration of100μg/μL; the group ofPBS inoculated with1×PBS (pH7.2). Bilateral tibialis anterior muscle injectionsmethod was used to immune the mice and100μL for each mouse. Immunization wasmade every two weeks from the first immunization and total3times. Then to screenthe immune effect groups and the sub-libraries that contains the protective antigengene all immunized mice were detected by specific antibodies in serum, the relativerate of protection, detection of cellular immune responses. The result showed the group1of genome-wide expression sub-library had a better effect immunization, whichproved that the library containing protective genes and lay the foundation for furtherscreening of the protective antigen gene contribute to the molecular levelunderstanding of avian Pasteurella multocida and search for fowl cholera genes. At thesame time, lay the foundation of developing the cheap, efficient vaccine against avianPasteurella multocida. The recombinant plasmids randomly selected from the library1were analyzed by the methods of restriction enzyme digestion, sequencing and theresults showed that the gene was a known pilus genes ptfa. The gene expressed animmuno-protein after the introduction of prokaryotic expression vector for inducedexpression which made good bedding for further in-depth development of the vaccine. |
PDF Full Text Request