腺病毒载体介导gag基因在小鼠体内的生物分布

目的研究腺病毒载体介导gag基因在C57BL/6小鼠体内的生物分布。方法经C57BL/6小鼠左后肢股四头肌部位单次注射0.05ml Ad-gag,于给药后不同时间摘取组织脏器,采用Taqman探针实时荧光定量PCR法,以小鼠β-actin基因作为内参,外标绝对定量法分别定量样本中gag和β-actin基因拷贝数。计算每104个小鼠细胞中gag基因的拷贝数,比较各脏器的分布数量。结果外标绝对定量法精密性良好。gag基因主要分布在注射位点、淋巴结、脾脏和肝脏,其他脏器没有分布。给药后3d,gag基因在靶器官分布达峰值,注射位点分布最多,伴随时间延长逐渐减少。结论Ad-gag疫苗在体内不会蓄积并产生全身的特别是生殖系统毒性。本研究可作为重要的安全性试验数据,支持药物在临床应用。

Biodistribution of gag Gene Transferred by Adenovirus Vector in Mice

Objective To study the biodistribution of gag gene transferred by adenovirus vector(Ad-gag)in mice.Methods Inject i.m.C57BL / 6 mice with 0.05 ml of Ad-gag and take tissues and organs on various days after injection for determination of copy numbers of gag and β-actin genes by real-time fluorescent quantitative PCR with Taqman probe,using mouse β-actin gene as internal reference.Calculate the copy number of gag gene in every 104 murine cells and compare the biodistribution of the gene in various or-gans.Results The external standard absolute quantification method used showed good precision.The result showed that gag gene was mainly distributed in injection site,lymph node,spleen and liver,while showed no distribution in other organs.The biodistribu-tion of gag gene in target organs reached a peak value 3 d after injetion,most of which were in injection site,and decreased as time goes on.Conclusion It is deduced that Ad-gag vaccine can not accumulate in vivo and cause systemic toxicity especially that in re-productive system.The study supports the clinical application of Ad-gag by providing important data of safety test.