摘要
目的采用活体成像技术快速评价DNA疫苗载体在小鼠体内的表达,为DNA疫苗载体的应用和改造提供参考。方法分别构建含有萤火虫荧光素酶基因(luciferase)及HIV-1CN54Gag基因DNA,且携带不同转录调控元件(posttranscriptional regulatory element)HPRE/WPRE的疫苗载体(p DRVI1.0Luc、p DRVI1.0HLuc、p DRVI1.0WLuc、p DRVI1.0Gag、p DRVI1.0HGag和p DRVI1.0WGag),均经后肢胫骨前肌注射BALB/c小鼠,20μg/只。p DRVI-1.0Luc、p DRVI1.0HLuc和p DRVI1.0WLuc组仅免疫1次,于免疫后第1、7、14天采用活体成像技术检测小鼠体内荧光表达强度;p DRVI1.0Gag、p DRVI1.0HGag和p DRVI1.0WGag组分别于第0、2、4周进行免疫,于末次免疫后第2周进行体液(ELISA法)及细胞免疫检测(ELISPOT法)。结果 p DRVI1.0Luc、p DRVI1.0HLuc、p DRVI1.0WLuc组小鼠于免疫后第1及7天,体内荧光均局限分布于注射部位,未出现扩散转移的现象,且p DRVI1.0HLuc质粒发光强度最强(P<0.05),免疫第14天各组均未检测到明显的荧光素酶表达;p DRVI1.0HGag组小鼠血清中抗Gag蛋白特异性抗体水平和Gag蛋白特异的分泌IFNγ的脾淋巴细胞数均明显高于p DRVI1.0Gag和p DRVI1.0WGag组(P<0.05)。结论活体成像技术可用于快速评价DNA疫苗载体在小鼠体内的表达,为DNA疫苗载体改造及免疫策略的优化提供了一个良好的研究平台。
Objective To rapidly evaluate the expression of DNA vaccine vector and provide a reference for the application and modification of DNA vaccine vector. Methods Luciferase and HIV-1 GN54 Gag coding sequence were digested with SalⅠand Eco RV and inserted into the Sal Ⅰ and Eco RV digested pDRVI1. 0, pDRVI1. 0H, pDRVI1. 0W vectors,producing DNA vaccines pDRVI1. 0Luc, pDRVI1. 0HLuc, pDRVI1. 0WLuc, pDRVI1. 0Gag, pDRVI1. 0HGag and pDRVI1. 0WGag, carrying posttranscriptional regulatory element HPRE / WPRE, respectively. BALB / c mice were immunized i. m. with the DNA vaccines respectively, 20 μg for each. The mice in pDRVI1. 0 luc, pDRVI1. 0HLuc and pDRVI1. 0Wluc groups were immunized once, of which the intensity of luciferase gene was analyzed by in vivo bioluminescence imaging technology on days 1, 7 and 14 after immunization. However, the mice in pDRVI1. 0 Gag, pDRVI1. 0HGag and pDRVI1. 0WGag groups were immunized for 3 times at weeks 0, 2 and 4, of which the humoral and cellular immunities were determined at week 2 after the last immunization by ELISA and ELISPOT assay. Results The fluorescence in mice of pDRVI1. 0Luc, pDRVI1. 0HLuc and pDRVI1. 0WLuc groups on days 1 and 7 after immunization were distributed evenly in injection site, without diffusion or transfer, of which the intensity in pDRVI1. 0 HLuc group was the highest（P〈0. 05）. No obvious expressions of luciferase were observed in various groups on day 14 after immunization.However, Gag-specific antibody levels and Gag-specific IFNγ secreting spleen lymphocyte counts in pDRVI1. 0HGag group were significantly higher than those in pDRVI1. 0Gag and pDRVI1. 0WGag groups（P〈0. 05）. Conclusion Bioluminescence imaging technology might be used for rapid evaluation of expression of DNA vaccine vector in mice, which provided a useful tool for modification of DNA vaccine vector and optimization of immune strategy.
出处
《中国生物制品学杂志》
CAS
CSCD
2016年第8期870-874,共5页
Chinese Journal of Biologicals
基金
中国医学科学院医学生物学研究所科技计划项目(IMB2013ZD01)
关键词
活体成像
DNA疫苗
Bioluminescence imagine
DNA vaccine
