重庆麻鸭乙型肝炎病毒基因组克隆及序列分析

目的 克隆重庆麻鸭乙型肝炎病毒(DHBV)全基因组并进行序列分析。方法 从自然感染DHBV的重庆麻鸭血清中提取DHBV基因组DNA,以聚台酶链反应扩增DHBV基因组DNA,克隆人PGEM—T载体,鉴定后行测序分析。结果 序列分析表明,重庆麻鸭HBV基因组全长3024 bp,由P、S/PreS及PreC/C基因组成。该DHBV株与GenBank中公布的HPUS5CG(M32990)同源性最高(94.9％),与DHBVCG(X74623)同源性最低(89.8％),其各个开放读码框起止位点与HPUGA相同,DNA和蛋白质序列中含有与基因调控和蛋白质功能相关的保守区。结论 成功克隆重庆麻鸭HBV全基因组,序列分析为进一步研究提供了有益的信息。

Sequence analysis of cloned duck hepatitis B virus genome from a Chongqing brown duck

Objective To clone and analyze duck hepatitis B virus genome from Chongqing brown duck. Methods Duck hepatitis B virus (DHBV) DNA extracted from a Chongqing brown duck was amplified by PCR and cloned into PGEM-T vector using T-A clone method. The sequence of this DHBV genome was analyzed with some softwares after identified. Results The duck hepatitis B virus genome from Chongqing brown duck (DHBVcq), which was 3 024 nucleotides long, contained three ORFs whose onset and end nucleotides were in accord with those of HPUGA, encoding P, PreC/C and PreS/S protein respectively. Comparison of this strain with other DHBV reported in GenBank showed that the homology of DHBVcq and M32990 got the highest score of 94.9% at nucleotide level, while DHBVcq and DHBVCG got the least (89.8%). Most of the conserved regulation nucleotides and amino acids sequence found in other DHBV were also identified in DHBVcq. The 6 region of DHBVcq ,which was important for encapsidation of pgRNA and synthesis of minus-strand DNA, differed from that of most other DHBV strains, forming a stem-loop conformation with a three-nucleotides upper stem rather than a common nine-nucleotides one in free status. Conclusion The successful clone and analysis of DHBVcq provide further studies with helpful information.