用人MHC转基因小鼠模型鉴定高致病禽流感病毒核蛋白CTL表位

Identification of CTL Epitopes in Nucleoprotein of H5N1 Influenza Virus by Using HLA Transgenic Mice

目的:筛选高致病禽流感病毒核蛋白(NP)中可用于高致病禽流感病毒感染检测或疫苗设计的CTL表位,为评价疫苗接种效果和开发新型疫苗奠定基础。方法:根据NCBI公布的NP的核苷酸序列设计特异性引物,以2006年深圳株高致病禽流感H5N1病人分离的病毒cDNA为模板扩增NP全长基因(1500 bp)并测序。通过生物信息学方法,预测NP氨基酸序列中潜在的HLA-A觹0201限制性表位。构建重组pJW4303-NP核酸疫苗并肌肉免疫HLA-A2/DP4转基因小鼠,利用ELISPOT法筛选特异性CTL表位。结果:克隆了2006年深圳株高致病禽流感NP基因,构建的重组pJW4303-NP核酸疫苗能在体外COS-7细胞中表达,免疫小鼠后能引起小鼠产生特异性的体液免疫和细胞免疫。结论:生物信息学和转基因小鼠模型筛选相结合的方法,能用于高致病性禽流感核蛋白CTL表位的筛选。

Objective： To identify CTL epitopes in nucleoprotein（NP） of H5N1 influenza virus.Methods： The cDNA of H5N1 influenza virus（A / Shenzhen / 406H / 06） and specific primers were used to amplify the NP gene.Af-ter sequencing,NP gene was subcloned into vector pJW4303 for immunization.We screened NP protein by bioin-formatics to predict potential HLA-A2 restricted epitopes and synthesized 10 top score epitopes.Subsequently,HLA humanized mice were immunized by pJW4303-NP plasmid and ELISPOT assay was used to observe specific CTL response.Results： pJW4303-NP recombinant plasmid was successfully constructed.After DNA immunization,signif-icant humoral and cellular responses could be detected in HLA transgenic mice.Conclusion： The method that combined bioinformatics and HLA transgenic mice can be applied to identify the CTL epitopes of H5N1.