改进的锚定反转录PCR法克隆中国人庚型肝炎病毒3′末端基因

• 作 者: 陈薇 黄策 徐静 付玲 王海涛
• 机 构: 军事医学科学院微生物流行病研究所
• 出 处: 解放军医学杂志
• 发表日期: 1997年06期
• 关键词: 庚型肝炎病毒 反转录 基因克隆 聚合酶链反应 锚定 献血员 病毒基因组 序列分析结果 基因片段 阅读框架

应用改进的锚定反转录ＰＣＲ（ａｎｃｈｏｒｅｄＲＴ－ＰＣＲ）法，在总ＲＮＡ的３′端“加尾”，Ｏｌｉｇｏｔｅｘ提纯ｐｏｌｙ（Ａ）＋ｍＲＮＡ，Ｏｌｉｇｏｄ（Ｔ）－ａｎｃｈｏｒ引物合成第１链ｃＤＮＡ，ａｎｃｈｏｒ引物及特异引物进行“半巢式”ＰＣＲ，从献血员血清中扩增出ＨＧＶ３′末端基因片段。序列分析结果表明：此中国株ＨＧＶ３′末端基因序列与美国株存在较大差异，但阅读框架相似。用此方法克隆单链ＲＮＡ病毒基因组的３′末端。由于许多动物病毒和９０％以上的植物病毒均为单链ＲＮＡ基因组，故该技术也可应用于其它病毒基因组未知末端的基因克隆。

The first strand cDNA and anchor were synthesized by modified anchored RT-PCR (PCR) with the Oligotex purified poly (A) + mRNA and Oligod (T) -anchor primers at the 3 ’ Primers and specific primers were subjected to “semi-nested” PCR to amplify the HGV 3 ’end gene fragment from the blood donors’ serum. The results of sequence analysis showed that the sequence of HGV 3 ’end gene in China was quite different from that of the U.S. strain, but the reading frame was similar. This method was used to clone the 3 ’end of the single-stranded RNA virus genome. Since many animal viruses and more than 90% of plant viruses are single-stranded RNA genomes, this technique can also be applied to gene clones at unknown ends of other viral genomes.