HBV(ayw)转基因小鼠HBV复制中间体的检测  

Detection of HBV replicated intermediates in the liver of HBV(ayw) transgenic mice

作　　者：陈阳述 尤玉琴 苏蔚 易学瑞 袁有成 陈文吟 张锋 孔祥平 

机构地区：[1]解放军458医院全军肝病中心、全军转基因动物重点实验室,广州510600

出　　处：《解放军医学杂志》2011年第9期959-961,共3页Medical Journal of Chinese People's Liberation Army

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(2008ZX10002-011)

摘　　要：目的建立稳定、高灵敏度化学发光Southern blotting检测系统,检测HBV(ayw)转基因小鼠肝脏及其他组织的HBV复制水平。方法采用地高辛标记HBV探针,利用pTHBV1047质粒检测杂交系统的灵敏度及重复性;提取第27代HBV(ayw)转基因小鼠肝、肾、脾和肌肉组织总DNA,经HindⅢ酶切后电泳、转膜,利用高灵敏度化学发光杂交Southern blotting检测各组织中HBV复制中间体的水平。结果地高辛标记的化学发光检测系统灵敏度达0.1pg,且杂交系统稳定可靠。HBV(ayw)转基因小鼠基因组DNA中检测到整合的外源HBV DNA,但低于HepG2.2.15细胞的HBV复制水平。肝、肾、脾和肌肉组织中都有HBV复制中间体存在,以肝组织中含量最高。结论第27代HBV(ayw)转基因小鼠染色体含有稳定的HBV基因整合,为复制型HBV转基因小鼠。Objective To establish highly sensitive and stable chemiluminescent Southern blotting system,and to detect the replication levels of HBV DNA in liver,kidney,spleen and muscles of HBV（ayw） transgenic mice.Methods The HBV probe was labeled with digoxigenin with PCR,and the sensitivity and reproducibility of hybridization system were detected by pTHBV1047.The total genomic DNA was extracted from the tissues of HBV transgenic mice and digested with Hind Ⅲ for 18 hours.After eletrophoresis,the digested DNA was transferred to nylon membrane.The replicated intermediates in the liver and other tissues of HBV transgenic mice were detected by chemiluminescent hybridization Southern blotting system.Results The sensitivity of the detection system labeled with digoxigenin was about 0.1pg,and the hybridization system was stable and reliable.Both the integrated transgene of HBV DNA and the HBV replicated intermediates were detected in the liver of HBV transgenic mice,but the level of HBV replication was lower than that in HepG2.2.15 cell line.HBV DNA replication also existed in kidney,spleen and muscles,but the replicated levels in these tissues were lower than that in the liver.Conclusions The 27th generation of HBV（ayw） transgenic mice is replicated in HBV transgenic mice with HBV gene stably integrated in the genome thereof.

关 键 词：小鼠 转基因 肝炎病毒 乙型 双杂交系统技术 

分 类 号：R512.62[医药卫生—内科学]