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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 in vivo by using live imaging technology and to provide a reference for the application and modification of DNA vaccine vector. Methods The vaccine vector (p DRVI1.0Luc, p DRVI1.0HLuc, p DRVI1) containing firefly luciferase gene and HIV-1CN54Gag gene DNA and carrying different transcriptional regulatory elements (HPRE / WPRE) was constructed. 0WLuc, pDRVI1.0Gag, pDRVI1.0HGag and pDRVI1.0WGag) were injected into BALB / c mice via tibialis anterior hindlimb at 20μg / mouse. p DRVI-1.0Luc, p DRVI1.0HLuc and p DRVI1.0WLuc were immunized only once, and the fluorescence intensity of the mice was detected by live imaging on days 1, 7 and 14 after immunization. p DRVI1.0Gag, p DRVI1 .0HGag and p DRVI1.0WGag groups were immunized at weeks 0, 2 and 4 respectively. Body fluid (ELISPOT) and cellular immunity (ELISA) were performed at the second week after the last immunization. RESULTS: Fluorescence in p DRVI1.0Luc, p DRVI1.0HLuc and p DRVI1.0WLuc mice was restricted to the injection site on days 1 and 7 after immunization. No proliferation and metastasis occurred in the pDRVI1.0Luc, (P <0.05). No significant luciferase expression was detected in each group on the 14th day after immunization. The level of anti-Gag protein-specific antibody in serum of p DRVI1.0HGag group was significantly higher than that of Gag protein-specific IFNγ secreting The numbers of splenic lymphocytes were significantly higher than those of p DRVI1.0Gag and p DRVI1.0WGag groups (P <0.05). Conclusion Live-imaging technique can be used to rapidly evaluate the expression of DNA vaccine vector in mice. It provides a good platform for DNA vaccine vector rebuilding and immunization strategy optimization.