EIAV反式激活蛋白(TAT)基因的分子克隆与序列测定

Cloning and Sequencing Equine Infectious Anemia Virus Trans-activating Protein

以马传染性贫血病毒 (EIAV)疫苗毒感染驴巨噬细胞培养物 ,在病毒繁殖期提取细胞总 RNA,反转录后使用特异引物扩增病毒基因组拼接产物。将扩增产物克隆、测序并与基因组全序列比较 ,确定了编码 EIAV反式激活蛋白(TAT)的转录产物及阅读框架 ,发现至少有 2种拼接产物编码 TAT。比较 2种阅读框架 ,以保守序列作为模板扩增得到完整的编码 EIAV反式激活蛋白的基因。

The donkey macrophage culture was infected with donkey leukocyte attenuated EIAV.At the early stage of virus replication the total RNA was extracted from infected cells.After reverse transcription,polymerase chain reaction was performed with EIAV specific primers.Then PCR products were cloned in the pUC19 plasmid vector and the inserts were sequenced.Two clones encoding TAT were obtained in this study.ORF encoding TAT and splicing sites were also determined.The whole sequence of tat gene has been obtained by further polymerase chain reaction during which more conservative sequence was used as template.