AIM:To design and construct an exogenous multiple epitopeof helper T lymphocytes(HTL),and to evaluate its effecton anti-HBs response through DNA immunization.METHODS:Artificial HTL epitope,PADRE and four otherHTL epitopes from different proteins were linked togetherusing splicing by overlap extension to generate exogenousmultiple epitopes of HTL,MTE5.pcNTE5 and pcHB weregenerated by cloning NTE5 and fragments of HBV pre-S2/Sgene into mammalian expression plasmid pcDNA3.Fourchimeric plasmids were constructed by cloning NTE5 intothe region of pre-S2 gene(bam HI),5’ terminal of S gene(HincⅡ,Xba Ⅰ)and 3’ terminal of S gene(Acc Ⅰ)of pcHBrespectively.BALB/c mice were used in DNA immunizationof the recombinant plasmids.Anti-HBs was detected usingAbbott INx AUSAB test kits.RESULTS:The sequences of MTE5 and the 6 constructs ofrecombinant plasmids were confirmed to be correct byDNA sequencing.The anti-HBs response of the co-inoculation of pcHB and pcMTE5 was much higher thanthat of the inoculation of pcHB only(136.7±69.1 mlU/mLvs 27.6±17.3 mIU/mL,P<0.01,t=-6.56).Among the4 chimeric plasmids,only the plasmid in which MTE5 wasinserted into the pre-S2 region had good anti-HBs response(57.54±7.68 mIU/mL),and had no significant differencecompared with those of pcHB and the co-inoculation ofpcHB and pcMTE5.CONCLUSION:Exogenous multiple epitopes of HTL hadimmune enhancement when they were co-inoculated withpre-S2/S gene or inoculated in the chimeric form at a propersite of pre-S2/S gene of HBV.It might suggest that it waspossible to improve hepatitis B vaccine using exogenousmultiple epitopes of HTL.The antibody responses werevery low using DNA immunization in the study.Thus,theimmune enhancement effect of exogenous multiple epitopesof HTL has to be confirmed and the effect on overcomingthe drawback of the polymorphism of HLA Ⅱ antigens shouldalso be evaluated after these chimeric plasmids areexpressed in mammalian cell lines. AIM: To design and construct an exogenous multiple epitope of helper T lymphocytes (HTL), and to evaluate its effecton anti-HBs response through DNA immunization. METHODS: Artificial HTL epitopes, PADRE and four otherHTL epitopes from different proteins were linked together by splicing by overlap extension to generate exogenous multi-epitopes of HTL, MTE5.pcNTE5 and pcHB weregenerated by cloning NTE5 and fragments of HBV pre-S2 / Sgene into mammalian expression plasmid pcDNA3.Fourchimeric plasmids were constructed by cloning NTE5 intothe region of pre-S2 gene (bam HI) , 5 ’terminal of S gene (HincII, XbaI) and 3’ terminal of S gene (AccI) of pcHBly probabilistically.BALB / c mice were used in DNA immunization of the recombinant plasmids. Anti-HBs was detected using Abbott INx AUSAB test kits .RESULTS: The sequences of MTE5 and the 6 constructs of recombinant plasmids were confirmed to be correct by DNA sequencing. Anti-HBs response of the co-inoculation of pcHB and pcMTE5 was much higher thant of the ino The expression of pcHB only (136.7 ± 69.1 mlU / mL vs 27.6 ± 17.3 mIU / mL, P <0.01, t = -6.56) .Among the4 chimeric plasmids, only the plasmid in which MTE5 was inserted into the pre-S2 region had good anti- HBs response (57.54 ± 7.68 mIU / mL), and had no significant difference with those of pcHB and the co-inoculation of pcHB and pcMTE5.CONCLUSION: Exogenous multiple epitopes of HTL hadimmune enhancement when they were co-inoculated with pre-S2 / S gene or inoculated in the chimeric form at a propersite of pre-S2 / S gene of HBV. It might suggest that it was able to improve hepatitis B vaccine using exogenous multi-epitopes of HTL. The antibody reaction were very low using DNA immunization in the study. Thus, theimmune enhancement effect of exogenous multiple epitopes of HTL has to be confirmed and the effect on overcoming the drawback of the polymorphism of HLA Ⅱ antigens shouldalso be evaluated after these chimeric plasmids areexpressed in mammalian cell lines.