华支睾吸虫RPEF基因的克隆与序列分析

Cloning and Sequence Analysis of RNA Polymerase Ⅱ Elongation Factor (RPEF) Gene of Clonorchis sinensis

目的 构建华支睾吸虫成虫RNA聚合酶Ⅱ延长因子(RPEF)基因PET重组质粒,分析其编码序列。方法 根据RPEF基因已知序列设计一对引物,用PCR技术从华支睾吸虫质粒文库模板中扩增RPEF基因片段;将目的基因PCR产物和空质粒PET30a(+)同时用BamHⅠ和SalⅠ限制性内切酶双酶切,纯化回收后建立连接反应并转化大肠杆菌BL21。将构建的重组质粒PET30a(+)-RPEF经双酶切、PCR、测序鉴定后证明获得正确的重组克隆。结果 成功构建出RPEF基因原核重组质粒PET30a(+)-RPEF。序列分析表明,RPEF蛋白与鼠类RNApolymerase Ⅱ延长因子具有很高的同源性。结论 RPEF基因在华支睾吸虫基因表达调控机制中可能起重要作用。RPEF基因重组表达载体地PET30a(+)-RPEF的成功构建可为更深入地分析其基因功能、肝吸虫病药物靶标的筛选奠定基础。

Objective To construct a recombinant plasmid and analyse encoding sequence of RPEF gene of Clonorchis sinensis. Methods A pair of primers was designed according to the known sequence of RPEF gene. The RPEF gene fragment was amplified by PCR. After purification and digestion with BamH Ⅰ and Sal Ⅰ, the RPEF gene was ligated to a prokaryotic expression vector, pET30a(+). Recombinant pET30a(+)-RPEF was constructed and transformed into E. coli BL21. Positive recombinants were detected by PCR, digestion with restriction enzyme and sequencing. Results The recombinant plasmid pET30a(+)-RPEF was constructed successfully. The result of sequence analysis showed that the RPEF protein of Clonorchis sinensis showed 81% of homology in the overall amino acid sequences with the mouse RNA polymerase Ⅱ elongation factor. Conclusion The construction of pET30a(+) -RPEF is important to further research on Clonorchis sinensis RPEF gene as a candidate target of anti- Clonorchis sinensis drug.