日本血吸虫RNA聚合酶转录因子SⅢ-p15真核正反义表达载体的构建和鉴定

CONSTRUCTION OF SENSE AND ANTISENSE EUKARYOTIC RECOMBINANT PLASMIDS EXPRESSING S.j RNA POLYMERASE ⅡTRANSCRIPTION FACTOR SⅢ-P15

目的 构建日本血吸虫 RNA聚合酶 转录延长因子 S - p15真核正反义表达载体 ,为进一步鉴定该因子的生物学功能奠定基础。方法 采用表达序列标签法从日本血吸虫成虫 c DNA文库中获得了 1个 RNA聚合酶转录因子 (S - p15 )全长 c DNA序列 ,克隆及序列测定后 ,将该片段正向及反向插入到真核表达载体 pc DNA 3中。重组载体采用氨苄青霉素 L B培养基筛选、双酶切、PCR及测序鉴定。结果 经鉴定 ,S - p15 - pc DNA3真核正反义表达载体构建成功。结论 构建成功 S -p15 - pc DNA3真核正反义表达载体 ,为下一步鉴定其生物学功能奠定基础。

Objective To construct the sense and antisense eukaryotic recombinant plasmids expressing S.j novel gene RNA polymerase Ⅱ elongin factor SⅢ p15. Methods The full cDNA sequence of S.j RNA polymerase Ⅱtranscription elongin factor SⅢ p15 was obtained by EST strategy. The fragment was inserted into sense and antisense eukaryotic recombinant plasmids by digesting with restrictive enzymes and linking reactions. The positive clone was screened on LB plates containing ampicillin and identified by restrictive enzymes digestion, PCR amplification and sequencing. Results Through identification, sense and antisense S.j SⅢ p15 pcDNA3 were constructed successfully. Conclusion Sense and antisense S.j SⅢ p15 pcDNA3 were constructed successfully and the further research will be carried out to testify its biological function.