TT型病毒基因组的克隆与进化分析

CLONING AND PHYLOGENETIC ANALYSIS OF HEPATITIS-ASSOCIATED TT VIRUS SUBGENOME

利用PCR方法从输血传播性病毒 (transfusiontransmittedvirus,TTV)阳性标本中获得不同长度且重叠覆盖TTV基因组的DNA片段。将PCR扩增片段克隆到pT Adv载体中 ,筛选获得阳性克隆。DNA序列测定结果表明所克隆的片段为TTV基因组序列。利用DNA片段中特有的限制性内切酶位点将TTV的DNA片段首尾相连 ,得到近全长的基因组克隆 ,命名为TTV0 2 1。对TTV0 2 1的核酸序列进行分析 ,TTV0 2 1长 3472nt,存在 2个阅读框架ORF1和ORF2 ,分别编码 785和 1 46个氨基酸。将TTV0 2 1与其它已知的TTV基因组全序列进行了同源性比较 ,并进行进化分析。结果表明 ,TTV0 2 1序列与TTV分离株CHN2、BDH1的遗传距离较近 ,而与其它分离株相对较远。

Polymerase chain reaction (PCR) was utilized for the DNA amplification from transfusion transmitted virus (TTV) positive serum samples.Five TTV DNA fragments,overlapped about 90% of the genome,were amplified by long template PCR for the generation of TTV subgenome.Recombinant plasmids were obtained by directly inserting PCR products into pT\|Adv vector,and DNA sequence analyses showed they were TTV DNA fragments.By using specific restriction enzymes,five TTV DNA fragments were ligated into a TTV DNA subgenome clone and named as TTV021.TTV021 has been deposited in GenBank database with the accession number AF254410.The results of computer analyses showed that TTV021, 3472 nt long,contains two open reading frames (ORF1, 785 aa;ORF2, 146 aa).Identity alignments between TTV021 and other TTV isolates indicated several high conserved regions existed.Phylogenetic analysis of 356 nt from TTV021 suggested that the isolate has close evolutionary relationship with CHN1 (type 1a),but has far relation with other TTV isolates.