| Brucellosis is an animal sourced zoonosis severely threatening public health. After infection, animal's reproduction and growth performance decrease greatly, result huge economic losses. In China, Brucella suis Stain 2(B.suis S2) is mainly used to prevent brucellosis in cow and sheep; however, this vaccine cann't differentiate antibody of vaccine immunization from spontaneous infection, bringing difficulties to epidemiological investigation and clinical serological diagnosis of brucellosis.Foot and mouth disease(FMD) is an acute, febrile and highly contagious infectious disease in cow and sheep, etc. caused by foot and mouth disease virus(FMDV). VP1, the major structural protein of FMDV, is the critical factor for virus infection of cells. With binding site for cell receptors, VP1 gene is the first target antigen choice for the research and development of recombinant vaccine.In this study, live B.suis S2 extensively used in China was used as a parent strain, which utilizes suicide plasmid vector to substitute gene cassette of FMDV VP1 to wbo A gene of B.suis S2, and successfully constructed recombinant B.suis vaccine carrying FMDV VP1. First, genome of B.suis S2 was used as the template, considering that wbo A gene was missed in carrier segment with chloramphenicol resistance, vitalities of 6 nonrecombined segments were screened by in vitro viability test. Then the recombinant shuttle plasmid was constructed, which included upstream and downstream homologous sequences of wbo A gene, and sucrose negative screening gene. The plasmid was electrically transformed into competent cell of B.suis S2 strain, and ampicillin resistance gene(Amp+) and sucrose negative screening gene(Sac B-) were utilized to screen the recombinant. The positive recombinant was identified by PCR and sequencing, named S2?wbo A/VP1 strain, and then biologically characterized.(1) Phenotype identification: Heat agglutination test, acriflavin test and colony crystal violet stain test demonstrated that, phenotype of recombinant S2?wbo A /VP1 was a rough phenotype, suggesting that the phenotype of parent strain without wbo A gene changed from smooth type to rough type.(2) Safety evaluation: Different doses of recombinant were injected into mice, cow and sheep, and rectal temperature, food intake and pathological findings, etc were observed. All results suggested that recombinant is safe for mice.(3) Identification of VP1 gene and study on humoral immunity of FMD: prokaryotic expression was conducted for VP1 gene to prepare antiserum and recombinant for Western Blot analysis, which suggested VP1 gene was successfully expressed in recombinant, and it had immunoreactivity. VP1 gene was marked with digoxin probe to perform Southern hybridization, and the result suggested that compared to parent strain, VP1 gene successfully inserted into a genome copy of B.suis S2. Indirect ELISA confirmed that, compared with parent strain, VP1 antibody generated 14 days later in mice of recombinant inoculation group, and the antibody level tended to quickly increase after the 3rd inoculation.(4) Study on immunogenicity of Brucelosis: Agglutination test on recombinant with different phenotypes demonstrated that, recombinant showed positive response with rough phenotype strains of B.melitensis M111 and B.canine RM6/66, and negative response to other strains of smooth type.Survival time of recombinant in mice was investigated by weighing spleen and separating bacteria. The results suggested that, day 14 of inoculation, spleen weight of mice was higher in recombinant group than that in parent strain group, with significant difference compared to control group(P<0.05). On day 42, vaccine strain even did not result in splenomegaly in mice(P>0.05, compared with the control), and no bacterium was separated from spleen. ELISA method was used to investigate the change of antibody. The results suggested that recombinant antibody was detected 7 days after inoculation, significantly increased from day 7 to 14, continued to increase from day 14 to 28 at a slow rate, and tended to decrease till day 42. Cytokine test indicated that, mice inoculated with recombinant strain S2?wbo A/VP1 and parent strain B.suis S2 showed basically consistent cellular immune response, started to generate IFN-? since day 7 of inoculation. IFN-? value continued to increase from day 7 to 28, and tended to decrease after day 7. Mice challenge test suggested that, 1×109CFU/mouse could provide 100% protection to challenge by B.meltensis M28.Challenge test was performed with sheep, Bacterial separation results from spleen and inguinal lymph nodes suggested that the inoculation group at 1×1011 CFU/sheep held 80% of protection percentage, similar to that provided by 1×1010 CFU/sheep parent strain of B.suis S2(80%).Brucellosis and FMD are important infectious diseases of domestic animals including cow and sheep. As the carrier with characteristics of intracellular parasitism, Brucellosis vaccine can sufficiently mediate T cell immunity in the host. Therefore, live recombinant B.suis vaccine carrying VP1 gene constructed and identified in this study provides an alternative vaccine strain to realize double immunization against brucellosis and FMD. |
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