体内电穿孔在质粒介导的基因转移及DNA免疫中的应用  

Application of in vivo electroporation technology in plasmid mediated gene transfer and DNA vaccination

作　　者：张舟[1,2] 徐智勇[2] 陈佩[2] 周芳 张宪省[1] 刘勇[2] 邵一鸣 

机构地区：[1]山东农业大学作物生物学国家重点实验室,泰安271018 [2]中国疾病预防控制中心性病艾滋病预防控制中心传染病预防控制国家重点实验室,北京100050 [3]宁波新芝生物科技股份有限公司

出　　处：《中华微生物学和免疫学杂志》2010年第6期551-554,共4页Chinese Journal of Microbiology and Immunology

基　　金：“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项“十一五”课题（2008ZX1001-1010;2009ZX10004-713）

摘　　要：目的 研究体内电穿孔技术对于质粒DNA介导的体内基因表达以及HIV-1 Env DNA疫苗免疫效果的影响.方法 将携带荧光素酶Luciferase基因的表达质粒p1.0-Luc和携带HIV-1 CN54 env基因的DNA疫苗质粒p1.0-gp1455m通过单独肌肉注射或肌肉注射后加电穿孔两种不同方法 注射小鼠.用IVIS（R）活体成像系统实时检测Luciferase报告基因在体内的表达情况.用ELISA检测HIV-1 Env特异的抗体反应,用IFN-γ ELISPOT检测HIV-1 Env特异的T细胞免疫反应.结果 体内电穿孔技术可以显著提高Luciferase在小鼠体内的表达水平,幅度达35倍.HIV-1 Env DNA疫苗免疫结果 显示,8μg质粒剂量电穿孔途径诱导的体液和细胞免疫应答强于40μg质粒剂量单纯肌肉注射组;体内电穿孔途径免疫2次与单纯肌肉注射途径免疫3次诱导的体液和细胞免疫应答水平相当.结论 体内电穿孔技术可以大幅度提高报告基因在体内的表达水平和DNA疫苗的免疫应答.Objective To investigate the effects of in vivo electroporation on plasmid mediated reporter gene expression and immunogenicity of DNA vaccine. Methods Luciferase expression plasmid was administered intramuscularly to BALB/c mice at 8μg and 40μg dosage level through injection with or without eletroporation Luciferase expression level in murine muscle was detected by IVIS imaging system 24 h after injection. DNA vaccine plasmid p1.0-gp1455m carrying codon-optimized env gene of CN54 strain （ HIV-1 CRF07_BC） was administered to mice at dosages of 8μg and 40μg through the two approaches mentioned above. Mice were immunized at week 0,2 and 4. Env-specific immune responses were detected at two weeks post the second and the third vaccinations. Env-specific antibody immune responses were determined by ELISA. Euv-specific cellular immune responses were determined by IFN-γ ELISPOT. Results Luciferase expression level in murine muscle was significantly increased as much as 35 folds through in vivo eletroporation. Results of ELISA and ELISPOT revealed that in vivo eletroporation could significantly enhance both the humoral and cellular immune responses induced by DNA vaccination. The responses induced by electrodelivered p1.0-gp1455m at 8 μg dosage were better than those induced by simple intramuscular injection with 40 μg of plasmid DNA. On the other hand, 2 injections followed by electroporation elicited comparable level of humoral and cellular immune responses with those induced by 3 injections without electroporation. Conclusion In vivo electroporation was capable of enhancing both the plasmid-mediated gene expression and immunogenicity of DNA vaccine.

关 键 词：体内电穿孔 DNA疫苗 基因表达 免疫应答 

分 类 号：R392.1[医药卫生—免疫学]