乙肝病毒共价闭合环状DNA标准品制备及鉴定  

Preparation and identification of Hepatitis B virus covalently closed circle DNA standard

作　　者：郭永灿 王友强 张丹 邓冲 赵容 涂植光[2] 

机构地区：[1]西南医科大学附属中医医院检验科,四川泸州646000 [2]重庆医科大学检验医学院,重庆400016

出　　处：《现代预防医学》2018年第1期129-133,共5页Modern Preventive Medicine

基　　金：泸州市重点科技计划项目(项目编号:2014-S-49);西南医科大学自然科学基金项目(项目编号:2015-YJ028)

摘　　要：目的通过构建乙肝病毒(HBV)重组质粒来制备共价闭合环状DNA(cccDNA),为cccDNA定量检测提供合适的标准品。方法选择本院就诊的2名慢性乙肝患者,采取蛋白酶K消化法提取其血清病毒DNA,运用高保真聚合酶扩增HBV DNA,然后将扩增片段插入克隆载体构建HBV重组质粒。以重组质粒为模板制备环状DNA,经测序鉴定和PCR定量检测。结果 HBV DNA全长序列被成功扩增,并顺利构建重组质粒。2个重组质粒经内切酶消化均能得到预期的HBV DNA片段(约3.2 kb)和载体DNA片段(约2.7 kb);经测序鉴定,2个质粒分别为HBV B和C基因型,其插入序列保真性好,错配率低,每kb分别为1.24 bp和1.56 bp;以重组质粒为模板制备的2个环状DNA长度均约为2.1 kb,测序证实为HBV cccDNA;经定量检测,其纯度高,浓度分别为1.05×10^(10)IU/ml和6.19×10~9IU/ml。结论利用重组质粒方式制备的HBV cccDNA具有纯度高、获取量大等优点,可作为cccDNA定量检测和方法性能验证的标准品。Objective To prepare the covalently closed circle DNA（cccDNA） by constructing recombinant plasmids containing HBV complete genome, and to provide suitable standard material to validate the method performance for detecting cccDNA. Methods Two chronic hepatitis B （CHB） patients were recruited in our outpatient. HBV DNA was extracted from sera of CHB patients by using protease K digestion method, and HBV complete genome was amplified with high fidelity PCR. Then recombinant plasmid included HBV complete fragments were constructed using T - A cloning technique. Moreover, two different circular DNA was prepared based on the recombinant plasmid and identified using sequencing and real -time PCR. Results HBV DNA fragments were smoothly amplified with high fidelity PCR from the sera of CHB patients. Two DNA fragments（ about 3.2 kb ） were inserted into pMD18 - T and recombinant plasmids containing full - length HBV genome were constructed successfully. In addition, 2 target fragments were identified after digested using endonucleases （Hind Ⅲ）, and their lengths were 2. 7 kb and 3.2 kb respectively, as same as expected. After sequencing, two recombinant plasmids were confirmed as HBV B and C genotypes respectively. The HBV complete sequences had excellent fidelity and their mismatch ratios were 1.24 bp/kb and 1.56 bp/kb. By sequencing, the length of circle DNA fragments was both 2. 1 kb and confirmed as HBV eeeDNA. Its purity was high,and their concentration was 1.05 × 10^10 IU/ml and 6. 19 × 10^9 IU/ml respectively. Conclusion HBV recombinant plasmids and cccDNA standard are successfully constructed in vitro. Furthermore, cccDNA have an advantage in high purity and a large amount, and it could be used as standard materials for cceDNA quantitative detection and performance validation.

关 键 词：乙型肝炎病毒 全长基因组 重组质粒 共价闭合环状DNA 标准品 

分 类 号：R113[医药卫生—公共卫生与预防医学]