Immunocompetence And Immunoprotection Of A Protein Vaccine With Neisseria Surface Protein A Fused With B Subunit Of Escherichia Coli Heat-labile Enterotoxin In Mice

Objective:The aim is to analyze the immunocompetence and immunoprotection of theprokaryotic recombinant protein vaccine LTB-NspA in BALB/c mice throughintraperitoneal inoculation, and explore if the mucosal immunoadjuvant LTB couldimprove NspA in inducing more effective specific humoral and cellular immunity.Besides,we want to evaluate its immunoprotection in BALB/c mice and provide some experimentalfoundation for the effective serogroup B Neisseria meningitides protein vaccine.Methods:We amplified the LTB and NspA from the Escherichia coli H44815and the Neisseriameningitidis MC58，and the prokaryotic recombinant vectors pET-30a/ltb、pET-30a/nspaand pET-30a/ltb-nspa were constructed by PCR and SOE PCR，then the recombinantvectors were transformed into E.coli BL21to express the recombinant protein. Therecombinant protein were purified through Ni-NTA-Resin, identified by Western-blot. Thefemale BALB/c mice were intraperitoneal inoculated with the purified recombinant protein,then analyze the levels of NspA-specific IgG and kinds of IgG in serum, SIgA in genitaltract,IL-4and IFN-γ in supernatant of spleen lymphocytes.The proliferation of spleenlymphocyte was tested by CCK8,then analyze the survival rate of BALB/c mice thatinoculated with the recombinant protein vaccine LTB-NspA.Results:The recombinant prokaryotic vetors pET-30a/ltb、pET-30a/nspa and pET-30a/ltb-nspawere successfully constructed and identified by double-enzyme cleavage and sequencinganalysis;After optimizing the induced and expressed conditions of the recombinantprotein in the soluble state,the recombinant protein were purified by Ni-NTA-Resin andidentified by Western-blot; The female BALB/c mice were peritoneal injected with the purified recombinant protein;The levels of IgG and IgG2a in serum in LTB-NspA groupwhich was significantly higher than the NspA Group (P<0.05); At the week6,the levels ofIgG1in serum in LTB-NspA group which was significantly higher than the NspA Group(P<0.05); At the week2and4,the levels of IgG2b in serum in LTB-NspA group which wassignificantly higher than the NspA Group (P<0.05);At the week4and6,the levels of IgG3in serum and SIgA in genital tract in LTB-NspA group which was significantly higher thanthe NspA Group (P<0.05);And There is no difference between the PBS group and theLTB group(P>0.05).All the ratio of IgG2a/IgG1in LTB-NspA group was less than1;The levels of IL-4and INF-γwere （204.778±18.936）pg/mL and （129.210±18.621）pg/mL in LTB-NspAgroup which was significantly higher than the NspA Group (P<0.05);The stimulation indexof the splenic lymphocyte was significantly higher than those of the PBS and LTB controls(P<0.05). The bactericidal titer of the LTB-NspA Group (6W) was1:64.After MC58wasinjected into the BALB/c mice,the survival rate of the mice immuned with recombinantprotein LTB-NspA was90%that higher than the the mice immuned with recombinantprotein NspA (70%) in72hours,and the survival rate of the mice immuned with PBS or therecombinant protein LTB was0.Conclusion:1. The LTB-NspA protein vaccine could induce specific humoral and cellullarmmunologic responses, especially Th2humoral immunological response；And fusingthe mucosal adjuvant LTB with NspA could induce effective mucosal immuneresponse.2. The LTB-NspA protein vaccine that showed effective immunoprotection for theBALB/c mice infected by the serogroup B Neisseria meningitides.