Objective To prepare the oral vaccine of respiratory syncytial virus(RSV)based on Lactococcus lactis(L.lac-tis)induction expression system and analyze its immunogenicity.Methods The constructed plasmids pUC57-Pre F and pNZ8149 were digested with Nco Ⅰ and Kpn Ⅰ,respectively,and the digested products were ligated with T4 DNA ligase to obtain the recombinant plasmid pNZ8149-Pre F,which was electroporated into the competent L.lactis NZ3900,and then screened by lactose medium to obtain non-secretory recombinant L.lactis NZ3900/pNZ8149-Pre F.Using nisin A as the inducer,the expressed products were analyzed by Western blot and immunofluorescence labeling technique.Further,female BALB/c mice were randomly divided into PBS,L.lactis NZ3900/pNZ8149,and L.lactis NZ3900/pNZ8149-Pre F groups,with 15 mice in each group and oral administration 500 μL per mouse.The initial immunization was performed on the 1 d and 2 d,and the booster immunization was performed on the 16 d and 17 d.On the 14 d and 28 d,blood samples were taken from the mandible of mice and spleen cells were isolated.The levels of humoral and mucosal immune responses induced by recombinant L.lactis NZ3900/pNZ8149-Pre F were determined by ELISA,while the levels of cellular immune responses by ELISpot.Results The recombinant L.lactis NZ3900/pNZ8149-Pre F was constructed correctly as identified by PCR and sequencing.The antigen protein Pre F was specifically expressed in L.lactis NZ3900 with a relative molecular mass of about 50 000.Compared with PBS and L.lactis NZ3900/pNZ8149 groups,mice in L.lactis NZ3900/pNZ8149-Pre F group had significantly higher serum Pre F-specific IgG titers,specific secretory IgA antibody levels in intestinal lavage fluid,IFNγ and IL-4 secretion levels in spleen cells on the 14 d and 28 d after the initial immunization(t=-30.268--3.087,each P＜0.05).Conclusion The RSV oral vaccine constructed based on L.lactis induction expression system has strong immunogenicity,which provides a reference for the development of RSV mucosal vaccine,and further provides a new research strategy and method for the development of oral vaccines against other viruses or bacteria.
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