Cross-protection Of Enterovirus 71 Vaccine And Construction Of Its Gene Recombinant Mycobacterium Smegmatis

Objective: HFMD is a common infectious disease caused by more than twenty kinds of enteroviruses.At present,only Enterovirus 71(EV71)vaccine is listed.Given the high degree of homology of enteroviruses,cross-reactivity between enterovirus has been reported.Therefore,in this study,we firstly explored the cross-neutralization of EV71 inactivated vaccine against the pathogens of HFMD and confirmed the value of the EV71 vaccine against other enterovirus.Secondly,recombinant Mycobacterium smegmatis vaccine based on the EV71 neutralizing epitopes VP1-SP70 and VP2-141,which is of great importance for the development of a HFMD vaccine,was constructed.The vaccine delivery vector Mycobacterium smegmatis has the advantage of efficient Adjuvant.The value of Mycobacterium smegmatis in constructing HFMD vaccine was explored.Methods:1.To amplify and culture eighteen isolated enterovirus strains,and detect the virus titers.EV71 inactivated vaccine was used to immunize rats to prepare antiserum.The titers of neutralizing antibodies against EV71 and thecross-neutralizing antibodies against other enteroviruses in the antiserum was detected using neutralization test.Sequence of P1 regions of enterovirus amplified by PCR was analyzed using clustalx software.Phylogenetic trees were constructed by MEGA6 software.2.The HBc-E1/2 with the VP1-SP70 and VP2-141 amplified by PCR.The HBc-E1/2 was cloned into the shuttle plasmid PMV261.The recombinant shuttle expressing plasmid of PMV261-HBc-E1/2 was transformed into Mycobacterium smegmatis by electroporation.The expression of HBc-E1/2 in Mycobacterium smegmatis was confirmed by SDS-PAGE and Western Blot.Results:1.The neutralization test showed that the neutralizing titer against the EV71 in the antiserum of EV71 inactivated vaccine immunized rats was 1024.The antiserum had only a weak cross-neutralization effect on CA6,CA9,CB4,ECHO6,ECHO11,ECHO14,ECHO30 and ECHO33 isolates,in which the neutralizing titer against CA9 was 16.Phylogenetic analysis showed that most of enteroviruses with cross-neutralization effect were Enterovirus B.CA6 that belong to Enterovirus A had a cross-neutralization effect.2.The expression of HBc-E1/2 in mc2155: HBc-E1/2 was confirmed by SDS-PAGE and Western Blot.The target protein was 25 KD and 50KD(dimer),and both VP1-SP70 and VP2-141 epitopes were expressed efficiently.Conclusions:1.Antiserum of EV71 inactivated vaccine immunized rats showed cross-protection against some enteroviruses.Phylogenetic analysis showed no significant relationship between neutralizing titers and genetic relationship.2.Recombinant Mycobacterium smegmatis vaccine mc2155: HBc-E1/2expressing HBc-E1/2 was successfully constructed,which laid the foundation for the subsequent experiment of immunogenicity.