Study On Vaccine Of Recombinant Peptide And DNA Based On Avian Influenza Virus M2 And Mucosal Immunization

Avian influenza virus(AIV)can infect almost all wild bird and domesticpoultries and especially lead to the death of poultry.Severe economic loss for thepoultry industry was brought because of the AIV prevalence.In 1997,HSN1 subtypeof AIV was isolated from flu patient in Hong Kong,the subtype ultimately causedseveral patients dead,and it was the first time reported that H5N1 AIV overacrossedinterspecies barrier and infected human.AIV has become a threat to human andgreatly engage the attention of public health.Although the vaccine immunization wasone of the important methods to prevent AIV reoccurrence,it is very difficult todevelop ideal vaccine which can provide protection against all subtypes of AIV thathas high frequent antigenic variation of HA and NA gene.The M2 protein of AIV,apotential candidate antigen for avian influenza vaccine with cross-protection,is atransmembrane protein and high evolutionary conservative in different subtypes.Because AIV infected animals through the respiratory and digestive tract,it wasimportant that AIV vaccine could elicit animals mucosal and system responses.Heat-labile enterotoxin(LT)has been regarded as one of the most powerful mucosaladjuvant,which eliciting strong immunoresponse to co-administered antigens.Especially LTB,without LT toxin,hold mucosal adjuvant activity of LT.In this study,fused genes based on M2 and M2e with the large carrier molecular HBcAg wereconstructed.The vaccines of peptide and DNA based fused genes were obtained,andimmune effects and LTB mucosal adjuvant activity were primarily studiedsimultaneity.Following was the mainly research results:1)M gene of a strain AIV isolated from Fujian province was cloned by RT-PCR andsequenced.The results of phylogenetic analysis of M gene with other 10 strainsisolated from China including a strain from human in 1997 in Hong Kong showedthat there was significant different between Fujian strain and Hong Kong humanstrain in heredity.M2 gene was amplified from M and then theâ–³M2 was constructed by the deletion of transmembrane segment(26-49aa)M2 by OE-PCR.The fused gene of M2eHBc and M2eHBc+ were constructed by insertion of M2eepitope into the N-terminus or/and MIR of HBcAg.The protein ofâ–³M2,M2eHBc and M2eHBc+ were expressed by the optimization of codons systems,and the purified proteins could react with M2 antibody and H5,H9,H7 standardpositive serum,M2eHBc and M2eHBc+ with the ability of self-assembly of VLPparticle.The M2-antibodies came from mice vaccinate with the 3 proteinsrespectively by intramuscular,and the M2eHBc+ immune effect significantlysurpassedâ–³M2 and M2eHBc immune groups.2)The LTB gene was amplified from LT which cloned from Escherichia coli 195,and LTB protein expression and purity were performed.The purified LTB proteincould react with CT antibody and have the activity of GM1-ELISA.LTB gene wasinserted into the N-terminus of M2eHBc+,and then the fused protein,LBM2eHBc+,was expressed and purified.The purified LBM2eHBc+ proteinwith the ability of self-assembly of VLP particle and the activity of GM1-ELISAcould react with M2 antibody.3)M2eHBc+ protein immunization alone resulted in a poor systemic IgG andmucosal IgA response by intranasal and oral routes,and supplementation of LTBprotein resulted in substantial stimulation of the serum IgG level and in inductiona strong IgA response by intranasal route.The result demonstrated that nontoxicLTB provided a potential promising immunoadjuvant for application in AIVsubunit vaccine.The immune effect of LBM2eHBc+ protein in mice by mucosalroute was essentially the same as that of the M2eHBc+ supplement LTB,butsurpassed the latter in inducing mucosal IgA response.4)The pCDNA3-M2eHBc+ DNA vaccine was constructed by fused M2eHBc+ geneinto the eukaryotic expression vector pcDNA3.Western blot analysis wasemployed to confirm the fused gene expression in transferred 293 cell.SpecificM2 antibody and T cell proliferation was detected in the immunized mice byintramuscular injection route.5)pCDNA3-LBM2eHBc+ DNA vaccine or pCDNA3-M2eHBc+ DNA vaccine with pCDNA3-LTB as supplementment immunized mice by intramuscular route,whichcould significantly enhance T cell response and no significant effect on systemicIgG,and if only by intranasal route could significantly enhance mucosal IgAsecretion.6)The results,development of DNA prime-protein boost immunization strategyagainst AIV,showed that systemic IgG response were significantly amplified andT cell response were no significantly affected,but mucosal IgA secretion whichwere induced by DNA vaccine in intranasal route was significantly affected.7)The fused gene LBM2eHBc+ were inserted into pPIC9K,and then transformedinto GS 115.By PCR analysis,it was proved that the gene was integrated into theGS115 genome.The positive clone,induced with 1% methanol,expressed aprotein which was about 39 KDa,and it's antigenicity was analyzed by westernblotting.