内含子A提高分枝杆菌热休克蛋白65 DNA疫苗的免疫原性  

Involvement of intron A into eukaryotic expression vector to improve immunogenicity of mycobacterial heat-shock protein 65 DNA vaccine in mice

作　　者：吴娟[1] 马辉[1] 范小勇[1,2] 曲勍[1] 罗玉萍[2] Douglas B．Lowrie 

机构地区：[1]复旦大学附属上海市公共卫生临床中心科学研究部,上海201508 [2]南昌大学生命科学与食品工程学院

出　　处：《中华预防医学杂志》2012年第1期58-63,共6页Chinese Journal of Preventive Medicine

基　　金：国家自然科学基金（30901276,31170876）;国家“十二五”重大传染病专项（2012ZX10003-006,2012ZX10003-007）：上海市青年科技启明星计划（09QA1405200）衷心感谢上海交通大学第六人民医院郭盛博士对本实验数据统计分析的指导与帮助

摘　　要：目的探讨以内含子A增强外源抗原在真核细胞中的表达效率及其对DNA疫苗在小鼠中免疫原性的影响。方法以分枝杆菌热休克蛋白65（Hsp65）为模式抗原，将其分别克隆入含有内含子A和不含内含子A的真核表达载体pCMV4．0和pVAX1，将表达Hsp65的不同重组质粒瞬时转染人胚肾上皮细胞系293T并免疫接种BALB／c小鼠，应用ELISA分析其所诱导产生的抗原特异性抗体水平及亚类，并以酶联免疫斑点试验（ELISPOT）和胞内细胞因子染色技术（ICS）分析其诱导产生的细胞免疫应答反应。结果含有内含子A的重组表达质粒pCMV4．0hsp65较之不含内含子A的pVAX1hsp65在293T细胞中的抗原表达量更高，免疫小鼠6周后诱导产生的抗-Hsp65特异性总IgG抗体水平（3．76±0．23对3．15±0．22，P〈0．01）和IgG2a／IgG1比值（4．08±0．04对2．234-0．12，P〈0．01）也更高，差异有统计学意义；分泌γ-干扰素（IFN-γ）的CD4^＋T淋巴细胞频率[（2．0±0．058）％对（1．5±0．087）％，t=4．804，P〈0．01]和CD8^＋T淋巴细胞频率[（0．6±0．058）％对（1．0±0．115）％，t=3．098，P〈0．05]增加，差异有统计学意义，提示其诱导产生了增强的Thl型免疫应答。结论内含子A可增强分枝杆菌Hsp65在真核细胞中的表达效率并可提高DNA疫苗在小鼠中的免疫原性。Objective To explore the involvement of intron A into eukaryotic expression vector to improve antigen expression efficiency and enhance immunogenicity of DNA vaccine in mice. Methods As model antigen, the coding gene of mycobacterial Hsp65 was cloned into eukaryotic expression vector pCMV4. 0 with intron A involved and pVAX1 without intron A involved, respectively. The resulted recombinant expression vectors were transfected into 293T cells and were then injected into BALB/c mice as DNA vaccines. Anti-Hsp65 specific IgG and isotype were detected by ELISA and T cell immune response was analyzed by enzyme-linked immunosorbent spot （ ELISPOT ） assay and intracellular cytokine staining. Results Compared with non-intron A pVAXlhsp65, the recombinant plasmid pCMV4.0hsp65 involved with intron A pVAXlhsp65 caused higher expression level of Hsp65 in 293T cells, and enhanced Thl type immune response,which was defined as higher level of anti-Hsp65 specific total IgG level （3.76 ± 0.23 vs 3.15 ±. 22,P〈0.01） and IgG2a/IgG1 ratio （4.08 ±.04 vs 2.23 ±. 12,P〈0.01） and more IFN-γ-secreting CD4^＋ （ （2. 0 ± 0. 058） % vs （ 1.5 ± 0.087 ） %, t = 4. 804, P 〈 0. 01 ） and CD8^＋ （ （0. 6 ± 0. 058）% vs （ 1.0 ± 0. 115 ）%, t = 3. 098, P 〈 0.05 ） T lymphocytes. The difference showed statistical significance. Conclusion Intron A can improve the expression efficiency of mycobacterial Hsp65 antigen and enhance immunogenicity of DNA vaccine in mice when involved into eukaryotic expression vector.

关 键 词：内含子 疫苗 DNA 免疫 热休克蛋白质类 

分 类 号：R392[医药卫生—免疫学]