Construction And Immunological Study Of Brucella Canis Ghost Vaccine Strains

Brucella canis, with rough coccobacilli, is an important biotype of Brucella spp which notonly can infect a variety of dogs, but also can infect human beings and other animals. Caninebrucellosis is caused by Brucella canis that is a natural foci zoonosis, which mostly transmittedby contact infection. Animals are usually infected by digestive tract, sometimes done by brokenskin and mucous membranes and respiratory tract. The livestock infected by Brucella canis,with recessive carrier state, has no significant clinical symptoms. But it is characterized byinvasion and harm of the reproductive system, such as aboution, orchitis, epididymitis, and alsolymph node inflammation, arthritis and other symptoms.Bacterial ghosts (BGs) are empty cell envelopes that lack cytoplasm and nucleic acid,which are unable to reproduce. Natural bacterial ghosts, infected with E.coli phage PhiX174, areformed by themselves’ lysis. Man-made BGs technology is mainly depended on the products bycontrolled expression of lysis gene E from E.coli phage PhiX174, through its oligomerization,conformational changes and other biochemical reaction to form a40-200nm in diameter acrossmembrane channels from most Gram-negative bacteria, therefore resulting in BGs in thepenetration by losing cell cytoplasm, nucleic acid and other components. Due to non-proteindenaturation, BGs exactly retain the same structure and cell membrane-associated antigenproteins, without modifying any immunogenicity. Meanwhile, BGs as novel inactivatedvaccines are better than formalin-killed vaccines on immunogenicity and protective efficacy intheory.In this study, a broad-host expression plasmid pBBR1MCS-2-TLC, with temperaturesensitive functions, was constructed and then electrically transformed into Brucella canisRM6/66. It is successful to screen and get plasmid-type Brucella canis ghost vaccine strain,which appeared to lose plasmid and then did not grow in18th generation. However, in order toovercome the problem of plasmid genetic instability in recombinant Brucella canis strain, thisexperiment combined with homologous recombination technology, constructed temperaturesensitive lysis type suicide plasmid pBK-CMV-SacB-B0419-TLC1, and then inserted thetemperature sensitive lysis components into Brucella canis genome and screened by kanamycinresistance and sucrose flat. Finally, the geneme-type Brucella canis ghost vaccine strain waswell constructed, without resistance to avoid the presence of resistance gene, which had a good genetic stablility in30th generation.After that, this study analysised these Brucella canis ghost vaccine strains by bacteriolyticdynamics test. By optimizing inductive conditions, plasmid-type Brucella canis ghost vaccinestrain was incubated at28℃to anOD600of1.2, the temperature was subsequently raised to42℃until30hours later to prepared BGs with lysis rate of100%, which counted approximately5.30×1010/mL. Compared to geneme-type Brucella canis ghost vaccine strain, the culture wasincubated at28℃to an OD600of0.6, the temperature was subsequently raised to42℃until30hours later to prepared BGs with lysis rate of100%, which counted approximately4.70×109/mL.Electron microscopic observation Brucella canis ghosts showed that their cell contents were lostthrough the lysis channel and their cell envelopes retained the complete surface structure.Considering above, geneme-type Brucella canis ghost vaccine strain could be considered as acandidate vaccine strain, which was more suitable than plasmid-type Brucella canis ghostvaccine strain. In addition, this study explored that the lysis rate might be associated withexpression of lysis gene E, growth state of Brucella strains and their phenotypes.At last, in order to explore application value of Brucella canis ghosts to be novel vaccines,this experiment evaluated safety and immungenicity of two Brucella ghosts respectively. It wasshowed that these Brucella canis ghosts had good safety which did not contain any live bacteriaand had no toxic side effects on mice by observing the physiological state and spleen index andindex grams on the immunized mice. In addition, the results of antibody dynamic of the serumon immunized mice showed that Brucella canis ghosts could effectively generate specifichumoral immunity, also maintained stable antibody level for10weeks. Antibody levels ofPBCG and GBCG groups could maintain at least for8weeks, but compared to FKB and S2groups only did for3and6weeks respectively. Lymphocyte subsets analysis indicated that thePBCG and GBCG groups could induce Th1-type cellular immune response, and cytokinesdetection experiments in vitro showed that PBCG and GBCG groups significantly secretedhigher levels of IFN-γ, IL-2, IL-4, IL-10and IL-12than the control group did, levels of IFN-γ,IL-2of PBCG and GBCG groups were significantly higher than FKB group, level of IL-10ofPBCG and GBCG groups was significantly higher than S2and FKB groups. Moreover, therewere no differences between PBCG and GBCG groups, revealing that Brucella canis ghosts assimilar as S2vaccine could stimulate body to generate a cellular immune response. The resultsindicated that geneme-type Brucella canis ghosts could provide an efficient protective immunitywith immune protection unit of2.37, while utilizing Brucella canis RM6/66strains to poisonattack on immunized mice. Similary, geneme-type Brucella canis ghosts also provided partly protective immunity with unit of1.60against Brucella melitensis16M strains in immunizedmice.In summary, this study successfully constructed plasmid-type and geneme-type Brucellacanis ghost vaccine strains, prepared by inductive condition optimized surface structureBrucella canis ghosts, which had good safety and immunogenicity as novel vaccines and couldbe worth of promising prospects.