摘要
目的 制备特异性靶向的嵌合DNA疫苗，并探讨其诱导机体产生免疫应答的效果。方法 用基因重组技术构建细胞毒T淋巴细胞抗原4（CTLA4）与戊肝病毒抗原HEVE2嵌合基因的真核表达质粒。同时构建不带CTLA4的pHEVE2作为平行对照质粒，体外转染COS-7细胞，Western blotting法检测细胞培养上清表达并且 物；于BALB/c小鼠皮内注射，每隔2周1次，共免疫3次，100μg/次，于免疫的第3、5和10周ELISA检测抗体效价。结果 获得pHEVE2和pCTLA4-HEVE2嵌合基因真核表达质粒，测序证实各连接点阅读框序列正确；体外转染COS-7细胞，证明真核质粒以分泌形式表达；接种pCTLA4-HEVE2小鼠产生高滴度的特异性抗HEVE2抗体，比接种pHEVE2的对照组高50-100倍；pCTLA4-HEVE2诱导高水平的IgG2a,IgG2bTh1型抗体和IgG1Th2型抗体应答，pHEVE2则以IgG1Th2型抗体应答为主。结论 CTLA4-HEVE2嵌合DNA疫苗有效地增强动物对HEVE2抗原的免疫应答，为进一步研究抗原靶向性的嵌合DNA疫苗奠定基础。
Objective To designchimericDNAvaccinetargetedto antigen-presentingcells(APCs)withenhancedefficacyto induceimmunization.Methods TheplasmidcontainingthegeneencodingcytotoxicT-lymphocyteantigen4(CTLA4),a sur-facemoleculeon T cells,wasdirectlyfusedto thegenefragmentHEVE2codingforhepatitisE virusantigenby molecularen-gineeringtechnology.TheplasmidcontainingHEVE2genefragmentalonewasalsoconstructedto serveas controlandtrans-fectionof COS-7cellswiththe2resultantplasmidswasperformedrespectively,followedbyassayof theexpressionsof CTLA4-HEVE2fusionproteinandHEVE2proteininCOS-7cellsby wayof Westernblotting.BALB/cmicewereinjectedintracuta-neouslywiththeDNAvaccine(100μg)for3timesat2-weekintervals,andthetiterof anti-HEVE2totalIgGandIgGsub-classesweredeterminedby enzyme-linkedimmunosorbentassay(ELISA).Results Themammalianexpressionplasmidsof CTLA4-HEVE2fusionproteinor HEVE2proteinalonewerecloned.Theculturesupernantsof COS-7cellstransfectedwith theplasmidsshowedtheproductionof CTLA4-HEVE2andHEVE2proteins,whichweresecretedin theformof dimers.MiceimmunizedwithpCTLA2-HEVE2producedhighlevelsof specificanti-HEVE2totalIgGtiterswithIgG2a,IgG2band IgG1subclassespredominantintheserum,approximately50-to100-foldhigherthanthoseinmiceimmunizedwithpHEVE2,whoseserumcontainedpredominantlyIgG1.Conclusion CTLA4-HEVE2chimericvaccinestimulatesstrongimmunere-sponses inmice,makingitpossibleforfurtherexplorationintochimericDNAvaccinesthattargettheantigento APCs.
出处
《第一军医大学学报》
CSCD
北大核心
2002年第2期97-101,共5页
Journal of First Military Medical University
