HSV-2gD核酸疫苗的构建及免疫效果观察  

Construction of a recombinant eukaryotic expression vector containing HSV 2gD and its immune responses

作　　者：焦凤萍[1] 刘莉 王玉[1] 于爱莲[1] 于广福[1] 

机构地区：[1]泰山医学院病原学教研室,山东泰安271016 [2]城市疾病预防控制中心,山东聊城252000

出　　处：《细胞与分子免疫学杂志》2012年第5期458-461,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：山东省卫生厅资助课题(2007HZ037);山东省卫生厅资助课题(2011QW029);山东省自然科学基金(ZR2009CQ003)

摘　　要：目的:在前期筛选的HSV-2gD模拟抗原表位序列P6的基础上,以pcDNA3.1(-)为载体、IL-18为佐剂,构建P6与IL-18串联的重组表达质粒,并在真核细胞中表达。将重组质粒免疫小鼠,观察免疫效果。方法:采用串联简并引物PCR法构建重组质粒pcDNA3.1-IL18-P6和pcDNA3.1-IL18,以酶切、测序鉴定。将重组质粒分别转染CHO细胞进行瞬时表达,通过间接免疫荧光和Western blot法检测P6的表达情况。将构建的真核表达质粒肌内注射免疫接种BALB/c小鼠3次,每次间隔1周。末次免疫后1周眼眶静脉采血,ELISA法检测小鼠血清特异性抗体滴度、IFN-γ及IL-18含量;末次免疫后1个月,处死小鼠,无菌分离脾脏,MTT法测定脾淋巴细胞增殖率。结果:构建的串联重组质粒pcDNA3.1-IL18-P6和pcDNA3.1-IL18经酶切及测序显示基因序列完全正确,经间接免疫荧光、Western blot法检测可证实模拟抗原表位序列P6具有近似天然序列的生物学活性。重组核酸疫苗pcDNA3.1-IL-18-HSV P6免疫小鼠后可刺激血清特异性抗体的产生,诱导分泌较高水平的IFN-γ和IL-18。可促进小鼠脾淋巴细胞的增殖。结论:成功构建和表达了重组质粒pcD-NA3.1-IL18-P6,其能诱导较强的细胞免疫和体液免疫,从而为构建更加有效的HSV-2DNA疫苗奠定了良好的基础。AIM： To construct and express pcDNA3.1- IL-18-HSV P6 recombinant DNA vaccine, and to observe the immune responses of pcDNA3.1-IL-18-HSV P6 genetic vaccine. METHODS： Genes of 1=6 and IL-18 were cloned into the eukaryotic expression vector pcDNA3.1 （ - ） respectively, and confirmed by enzyme digestion and sequence analysis, the recombinant plasmid pcDNA3.1-IL-18-HSVP6 was trans- formed into CHO cells. The expressed protein was charac- terized by indirect immunofluorescence and Western blotting. The recombinant plasmid pcDNA3.1-IL-18-HSVP6 was used to inoculate BALB/c mice by intramuscular injec- tion for three times, once a week. One week after the last vaccination, the levels of specific IgG antibody, IFN-y and IL-18 were detected by ELISA; One month after the last vaccination, spleen cells of vaccinated mice were separated and proliferation of spleen lymphocytes were detected by Ml-r assay. RESULTS： Recombinant pcDNA3. 1-IL-18-HSV P6 plasmid was confirmed by enzyme digestion and DNA sequencing. After inoculated by pcDNA3. 1-IL-IS-HSV P6 vaccine, the mice could produce higher level of splenocytes proliferation and secrected higher level of IFN-γ, IL-18 and specific antibody. CONCLUSION： DNA vaccine of pcD- NA3. I-IL-18-HSV P6 has been successfully constructed, and it can effectively induce humoral and cellular immune responses, which prorided a basis for constructing new type of DNA vaccine.

关 键 词：单纯疱疹病毒 白细胞介素-18 模拟抗原表位 疫苗 

分 类 号：R392.12[医药卫生—免疫学]