Comparison Of Immune Effects Between Eukaryotic And Prokaryotic Expression Of Recombinant Omp10-Omp28-L7/L12 Subunits Of Brucella

Brucella,the pathogen of brucellosis,infects livestock as well as humans,posing a potential bioterror threat in endemic areas.Nowadays,some live attenuated vaccines of Brucella are used to immunize livestock.However,these vaccines are pathogenic for humans,which can provoke abortion when administered to pregnant livestock and induce antibodies in vaccinated livestock that interfere with the diagnosis of field infection.It is therefore crucial to improve the immunoprotective effects and safety of vaccines against Brucella.Currently,recombinant protein-based subunit vaccines have been considered promising alternatives for the next generation of safe and effective intervention against brucellosis.Here,we expressed the recombinant Omp10-Omp28-L7/L12 proteins of Brucella by eukaryotic and prokaryotic systems that were used as immunogens for evaluating immune response.In addition,our team found that Taishan pinus pollen polysaccharide?TPPPS?was used as an adjuvant to study its immunoregulatory effect on immunogen.The mice were divided into four groups and inoculated separately with Omp10-Omp28-L7/L12?P.pastoris expression products?,Omp10-Omp28-L7/L12?P.pastoris expression products?+TPPPS,Omp10-Omp28-L7/L12?E.coli expression products?and phosphate buffer?PBS?.Subsequently,we examined antibody levels,spleen lymphocyte proliferation,the number of CD₄⁺and CD₈⁺T cells,and cytokine secretion of mice.And evaluated the immune efficacy of the vaccines by challenge protection test.The immune efficacy of the respective vaccines was evaluated by protection against experimental challenge.Analysis of experimental results showed that the immune effects of Omp10-Omp28-L7/L12 recombinant protein inoculated groups were significantly higherthanthoseofthePBScontrolgroup.Moreover,therecombinant Omp10-Omp28-L7/L12 protein expressed in P.pastoris exhibited higher immunogenicity than that expressed in E.coli.Furthermore,TPPPS was proved to be a good immunopotentiator for the recombinant Omp10-Omp28-L7/L12 protein.Thus,the combination of recombinant Omp10-Omp28-L7/L12 subunit and TPPPS adjuvant shows potential as an effective Brucellosis vaccine.The designed Omp10-Omp28-L7/L12 fusion gene sequence was synthesized by GENEWIZ after codon optimization.And the fusion genes amplified by PCR were connected to the cloned plasmid pMD18-T vector,and the recombinant plasmid was transformed into E.coli DH5?strain to increase the yield.The correct pET-28a?+?-Omp10-Omp28-L7/L12 E.coli transformants and blank expression plasmid pET-28a?+?were cultured by IPTG with shaking at 37?for 6 hours.The bacterial solutions were centrifuged at 4?and the lysates of the bacteria were collected at 1,3,5,and 6 h post IPTG induction.At the same time,polyclonal antibodies against Omp10,Omp28 and L7/L12 were prepared as follow-up experimental materials.The correct pPIC9-Omp10-Omp28-L7/L12 transformants and blank expression plasmid pPIC9 were transformed into P.pastoris and induced by methanol.The culture supernatants were harvested through centrifugation at 24,48,72,and 96 h after methanol induction.It was identified by SDS-PAGE and Western blot analysis that a band with a molecular weight of 55.4 kDa was visualized in the protein gel chromatography,and the band was not found in the negative control group.It was observed that the band was consistent with the size of the target protein.Western blot analysis also showed bands of the same size as SDS-PAGE analysis.After the expression product was purified by nickel column,it was again subjected to SDS-PAGE gel electrophoresis,and only a target band having a molecular weight of 55.4 kDa was found in the gel chromatography.Theproteinvaccinewaspreparedbymixingthepurifiedrecombinant Omp10-Omp28-L7/L12 protein with the TPPPS extracted in the laboratory at a ratio of 1:1.A total of 120 5-6 weeks old BALB/c female mice were randomly divided into 4 groups of 30animals each and housed under the same environmental conditions.All mice in the I-IV group were subcutaneously inoculated with pure Omp10-Omp28-L7/L12?P.pastoris expression products?,Omp10-Omp28-L7/L12?P.pastoris expression products?+TPPPS,pure Omp10-Omp28-L7/L12?E.coli expression products?and PBS,and boosted on days 7 and 14after inoculation.At 0,14,28,42 and 56 dpv after inoculation,5 mice in each group were randomly selected for blood collection and the relevant immune indicators were evaluated by ELISA,flow cytometry and MTT methods,respectively.The following indicators were mainly tested:serum antibody titer,cytokine?IL-2,IFN-??levels,peripheral blood CD₄⁺and CD₈⁺levels,and T lymphocyte transformation rate.The immune effects of the vaccines were also evaluated by challenge protection tests.The results showed that the recombinant protein Omp10-Omp28-L7/L12 expressed by P.pastoris stimulated the body to produce specific antibodies with better immunoprotective effect than Omp10-Omp28-L7/L12 expressed by prokaryotic E.coli.TPPPS as an adjuvant significantly enhanced the body's immune response level.Our findings provide new prospects for improving the immune efficacy of the Brucella subunit vaccine.