实时定量PCR技术研究DNA疫苗在小鼠体内的生物分布  

Development and validation of quantitative PCR method for studying biodistribution of a HIV-PV DNA vaccine in mice

作　　者：付洁[1] 李丁[2] 夏绍友[3] 宋海峰[1] 董增祥[1] 陈方[1] 孙效[1] 欧伦[1] 汤仲明[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850 [2]第四军医大学免疫研究所,西安710032 [3]解放军总医院普通外科,北京100853

出　　处：《中国新药杂志》2009年第8期728-732,766,共6页Chinese Journal of New Drugs

基　　金：国家自然科学基金(30500625)

摘　　要：目的：建立并确证实时定量PCR（RT-PCR）定量检测生物基质中质粒DNA的方法,并应用于治疗性核酸疫苗HIV-PV的体内生物分布研究。方法：建立SYBR Green荧光定量PCR绝对定量方法（Q-PCR）,考察方法的特异性、灵敏度、精密度、准确度以及生物基质对PCR的抑制效应。小鼠尾静脉注射裸质粒DNA、小鼠尾静脉注射和灌胃2种途径免疫HIV-PV DNA疫苗,考察建立的Q-PCR方法对于不同形式的质粒DNA疫苗以及不同免疫方式的适用性。结果：Q-PCR方法可特异检测目标DNA,定量检测范围为10-10^7拷贝/100 ng基因组DNA（gDNA）,定量下限（LLOQ）为10拷贝/100 ng gDNA。批内和批间精密度分别〈11%和8%;由高至低4种浓度（10^7,10^4,100,10拷贝/100 ng gDNA）QC样品准确度分别为（1.57±0.02）%,（1.60±0.01）%,（2.20±0.02）%和（2.28±0.02）%。用Q-PCR方法成功得到不同形式DNA疫苗和不同免疫途径（尾静脉注射和灌胃HIV-PV疫苗）下该质粒DNA的生物分布模式。结论：利用RT-PCR技术,建立了符合DNA疫苗生物分布研究要求的定量检测生物基质中质粒DNA的方法学,并成功应用于不同质粒DNA疫苗形式和不同免疫途径下的DNA疫苗的生物分布研究。Objective：To develop and validate a quantitative real-time PCR （RT-PCR） method for plasmid DNA copies （PCN） determination in biological matrix. The method might be applied for investigating the distribution of a HIV-PV DNA vaccine in mice. Methods： SYBR green real-time PCR absolute quantitative （Q-PCR） methods were established. The specificity, sensitivity, precision, accuracy and the PCR inhibition by biological matrix were evaluated. The applicability of Q-PCR methodology was investigated by i. v. administration of naked plasmid pUMVC3-HIV-Gag vaccine, and i. v. and i. g. administration of HIV-PV DNA vaccine. Results： The specificity was well, and quantitative linear range was within 10 - 10^7 copies/100 ng genomic DNA（gDNA）. Lower limit of quantitation （LLOQ） was 10 copies/100 ng gDNA. Intra- and inter-variability was below 11% and 8% , respectively. The accuracy of quality control （QC） samples at 4 concentrations（10^7, 10^4, 100, 10 copies/100 ng gDNA） was （1.57 ±0.02）%, （1.60 ±0.01）%, （2.20 ±0.02）% and （2.28 ±0.02）%. The PCN changes were investigated by Q-PCR methods established in this study. Conclusion： Using real-time PCR, the method for PCN determination is well established in biological matrix according to the regulation of DNA vaccine biodistribution study, and the methodology is successfully applied in DNA vaccine biodistribution in mice despite of the form of DNA vaccine （naked or HIV-PV DNA vaccine） and the administration routes（i. v. and i. g. ）.

关 键 词：DNA疫苗 实时定量PCR 生物基质 绝对定量 生物分布 

分 类 号：R915[医药卫生—微生物与生化药学]