摘要
目的:扩增并克隆耐拉米夫定乙型肝炎病毒(HBV)变异株全基因组DNA,为构建含双体HBV YMDDDNA质粒和HBV YMDD变异株细胞模型及研究药物筛选奠定基础。方法:从一名耐拉米夫定的慢性乙型肝炎病人血清中提取HBV病毒DNA,用错配PCR结合限制性片段长度多态性(RFLP)进行变异分析,然后采用PCR方法在HBV负链开环缺口处及基因组中单一酶切点(SpeⅠ)处,设计两对引物从两个方向分别扩增1.15 kb和2.05 kb的单链及双链DNA区,利用基因重组技术,将两片连接后克隆至质粒pcDNA3.1-中,再进行DNA序列测定。结果:错配PCR结合RFLP分析证实该病毒存在YVDD变异,通过两段法克隆出HBV全基因组,经序列分析所得为HBV YMDD变异株全基因组。结论:成功克隆出HBV YMDD变异株全基因组。
Objective： To construct cell model of HBV YMDD strain and to screen the medicine against the disease associated with HBV YMDD. Methods： The full-length of HBV genome was cloned and identified from sera of the lamivudine-resistant HBV infected patient. Genome DNA of HBV was isolated from serum to analyse the mutation by mismatched PCR in combination with enzyme disgestion. Then PCR products of HBV genome were amplified separately from the single-stranded and double-stranded regions of the genome, and the products of PCR were cloned into plasmid and identified by DNA sequencing. Results： The two PCR products obtained were 1. 15 kb and 2.05 kb. And two sets of primers were designed according to the nick region and a single-cutting restriction enzyme site （Spe I ） of HBV molecule. After cloning the PCR products into a plasmid, the sequences of HBV genome were obtained. Through the method of DNA sequencing, the cloned HBV genome was confirmed as the HBV YMDD mutant strain. Conclusion： The fulllength genome of FIBV YMDD mutant strain had been cloned successfully.
出处
《武汉大学学报（医学版）》
CAS
2006年第1期5-8,16,共5页
Medical Journal of Wuhan University
基金
深圳市科技局资助项目(编号:200204185)
广东省中医药管理局资助项目(编号:402024)
