携带EBV-LMP1Δ基因的DNA疫苗和腺病毒疫苗联合免疫效果研究  

Construction of Epstein-Barr virus LMP1-deleted(LMP1Δ) DNA vaccine and a preliminary study on the effect of its combination immunization with adenovirus vaccine

作　　者：尉秀霞[1] 王湛[2] 周玲[2] 杜海军[2] 叶树清[2] 曾毅[2] 

机构地区：[1]北京市丰台区疾病预防控制中心,北京100071 [2]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京100052

出　　处：《中国病毒病杂志》2012年第4期250-254,共5页Chinese Journal of Viral Diseases

基　　金：国家高技术研究发展计划(863计划)资助项目(2006AA02A229;2012AA02A404)

摘　　要：目的构建携带EBV-LMP1Δ基因的DNA疫苗,并与携带LMP1Δ基因的重组非复制5型腺病毒疫苗联合免疫,观察在小鼠体内诱导LMP1Δ特异性细胞免疫应答的效果。方法用PCR方法获得去除癌基因的LMP1Δ基因,插入到pcDNA3.1(+)-His质粒载体中,构建携带LMP1Δ基因的DNA疫苗pcDNA-LMP1Δ。使用pcDNA-LMP1Δ单独或与携带LMP1Δ基因的非复制5型重组腺病毒疫苗Ad-LMP1Δ联合免疫Balb/C小鼠,末次免疫1周后应用IFN-γELISPOT方法检测小鼠脾淋巴细胞中LMP1Δ特异性细胞毒性T淋巴细胞(CTL)水平。结果构建成pcDNA-LMP1ΔDNA疫苗,酶切及测序证实LMP1Δ基因插入正确,在细胞中能有效表达LMP1Δ蛋白。pcDNA-LMP1Δ单独免疫Balb/C小鼠能够诱导产生LMP1Δ特异性的CTL应答;与Ad-LMP1Δ联合使用后,能够提高特异性细胞应答水平。结论构建的重组DNA疫苗pcDNA-LMP1Δ能够有效表达LMP1Δ蛋白,并能够在小鼠体内诱导出LMP1Δ特异性的CTL反应;与重组腺病毒疫苗联合应用,可以提高特异性CTL应答水平。Objective To construct a DNA vaccine that carries Epstein-Barr virus（EBV）latent membrane protein 1-deleted（LMP1Δ）gene,and immunize mice in combination with recombinant replication-deficient adenovirus type 5 vaccine carrying LMP1Δ,and observe the effect of LMP1Δ-specific cellular immune response induced in mice.Methods EBV LMP1Δ gene was obtained by PCR and inserted into plasmid pcDNA3.1（＋）-His to construct a DNA vaccine pcDNA-LMP1Δ.The Balb/C mouse CD8＋ epitopes specific to EBV LMP1Δ were screened using a synthesized 15-amino acid EBV LMP1Δ polypeptide pool.Balb/C mice were immunized（intramus-cular injection） with pcDNA-LMP1Δ alone,or in combination with a recombinant replication-deficient adenovirus type 5 vaccine that carries the EBV LMP1Δ gene as well.One week after the final immunization,the EBV LMP1Δ-specific CTL levels in the mouse spleen lymphocytes were analyzed using IFN-γ ELISPOT assay.Results The EBV LMP1Δ gene was correctly inserted into the pcDNA vector and the pcDNA-LMP1Δ DNA vaccine was constructed as confirmed by enzymatic digestion and DNA sequencing.The specific-EBV LMP1Δ protein expression was detected by immunostaining in human embryonic kidney 293 cells.A Balb/C mice CD8＋ epitope specific to LMP1Δ was found in the screening and immunization of Balb/C mice with pcDNA-LMP1Δ alone could induce LMP1Δ-specific CTL response;the specific CTL response could be improved when mice immunized in combination with the recombinant adenovirus vaccine.ConclusionsThe constructed DNA vaccine pcDNA-LMP1Δ could effectively express EBV LMP1Δ protein and induce the LMP1Δ-specific CTL response in mice.The specific CTL response could be improved when immunizing mice in combination with the recombinant adenovirus vaccine.

关 键 词：EB病毒 潜伏膜蛋白-1 DNA疫苗 

分 类 号：R392.12[医药卫生—免疫学]