【摘要】 目的　证实乙型肝炎病毒 (hepatitisBvirus,HBV)前基因组RNA在 4 5 8nt～ 130 8nt(nt,核苷酸 )之间可发生剪接并产生相应的基因组剪接变异体。方法　对 1株分离自慢性乙型肝炎患者血清的亚基因组HBVDNA(2 36 6bp)进行测序并以VectorNTI6 .0软件进行分析。将全基因组HBVDNA(32 15bp)理论上可能的剪接供体及受体内的核苷酸进行定点突变并将突变株转染HepG2细胞 ,以位于HBVDNA 4 5 8nt及 130 8nt两侧的特异引物检测转染后的细胞内HBV核心颗粒DNA ,并以相同引物检测 4 5 8nt～ 130 8nt发生缺失的HBVDNA在不同病程的乙型肝炎患者血清中的存在情况。结果2 36 6bpHBVDNA在 4 5 8nt～ 130 8nt之间发生缺失并符合GT AG剪接模式。 32 15bp全基因组HBVDNA在 4 5 9nt及 130 6nt分别发生G→A及A→C突变后均不能产生 4 5 8nt～ 130 8nt缺失。 4 5 8nt～130 8nt发生缺失的HBVDNA在慢性乙型肝炎、肝硬化及原发性肝细胞癌患者血清的检出率分别为80 %、75 %及 70 % ,显著高于HBV无症状携带者 10 %的检出率。结论　HBV前基因组RNA在 4 5 8nt～130 8nt之间可发生剪接并产生长度为 2 36 6bp的基因组剪接变异体。该类型基因组剪接变异体基因结构特点及在HBV相关性肝病中广泛存在提示它与HBV致病性密切相关。更多还原

【Abstract】 Objective To verify that spliced variant of hepatitis B virus genome can be generated by pregenomic RNA splicing ranging from 458nt to 1?308nt. Methods A subgenomic HBV DNA with 2?366bp isolated from a patient with chronic hepatitis B was sequenced and analyzed by Vector NTI6.0 software. Nucleotide within the theoretical splicing donor and acceptor in full length HBV genome mutated respectively and the mutants were used to transfect the HepG2 cells. Intracellular core particles were harvested and a pair of primer flanking the 458nt-1?308nt region was used to amplify the HBV DNA within the core particles. The same primers were also used to detect the presence of HBV DNA with the deletion in the region of 458nt-1?308nt in the serum from the patients with different clinical manifestations. Results 2?366bp HBV DNA showed deletion in the region spanning from 458nt to 1?308nt as compared to the full length counterpart and this type of deletion fitted with the GT-AG splicing mode. Either the point mutation from G to A in 459nt within the putative splicing donor or from A to C in 1?306nt within the putative splicing acceptor in 3?215bp full length HBV DNA vanished the 458nt-1?308nt deletion in HBV genome. 458nt-1?308nt deletion could be detected in the patients with chronic hepatitis, cirrhosis or hepatocellular carcinoma with the rate of 80%, 75% and 70% respectively, and they were much higher than 10% in asymptomatic carrier. Conclusions Spliced variant of hepatitis B virus genome with the length of 2?366bp was generated by HBV pregenomic RNA splicing ranging from 458nt to 1?308nt. Characteristics of gene organization and prevalence in HBV-related diseases indicated that such type of spliced variant may be closely related to pathogenesis of HBV.更多还原