乙型肝炎病毒全基因克隆及序列分析发现一D/C基因型重组株

A genotype D/C recombination strain of hepatitis B virus isolated from HBV carrier in China

目的 构建含有adr、adw、ayw 3个血清型的HBV全基因组质粒 ,并进行测序及序列分析。方法 从HBsAg携带者的血清中扩增出S区克隆 ,筛选出adr、adw、ayw血清型的血清 ,然后扩增HBV的全基因组 ,对扩增产物进行克隆测序。利用DNAStar的MegAlign ,Phylogenetictree对HBV全基因序列以及分段序列进行比较和进化树分析。结果 构建了含有adw、adr、ayw 3种血清型的HBV全基因组质粒 ,代码分别为 :H1、H2、H3,对 3株完成序列测定 ,按照S区核苷酸顺序划分基因型分别为B、C、D ,全序列分型显示H1、H2为B、C基因型 ,与按S区的分型一致 ,但H3为C基因型 ,与按S区的分型不同 ,进一步的序列分析表明H3为一株异常的基因型 ,按照S区和preS2区分型为D基因型 ,但是全基因组 C P X区的进化树分析 ,H3株均位于C基因型 ,提示该异常的基因型可能是由于D C基因型间基因重组引起 ,对重组位点进行了分析 :3181nt～ 10nt、799～ 834nt为可能的重组位点所在区。结论 H3病毒株的发现进一步证明了HBV基因型间的基因重组 ,但澄清该问题需要在HBV感染的高流行区进一步的积累HBV异常基因型的全序列 ,以及发现新的基因型资料。

Objective To clone and sequence a full-length HBV genomes from the blood of chronic HBV carriers. Methods To extract HBV DNA from the blood of chronic HBV carriers. Full-length HBV genomes were amplified with long PCR. The amplicons were cloned into pGEM-Teasy vector and sequenced by the chain termination method with overlapping primers. The sequences were analyzed with DNAStar, ClustalW method MegAlign and Phylogenetic tree software. Results The H3 strain is an aberrant genotype. In phylogenetic tree analysis it was clustered with genotype D based on S-ORF and preS2, but it was clustered with genotype C based on full-length genome, P-ORF, C-ORF and X-ORF. Phylogenetic tree analysis of these segments suggested the recombination between genotype C and genotype D. The possible recombination sites are 3181-10nt and 799-834nt. Conclusion This study demonstrate the recombination between genotype C and D. Clarifying recombination events in HBV requires further study of putative new genotype and accumulation of the aberrant genotype HBV database especially in HBV high prevalent regions.