促血管生成素-2过表达重组腺病毒载体的构建

Construction of recombinant adenovirus vectors overexpressing angiopoietin-2

目的构建小鼠促血管生成素-2(Ang-2)基因过表达重组腺病毒载体。方法化学合成小鼠Ang-2编码序列,经PCR扩增、鉴定后连接至穿梭质粒。将含目的基因的重组质粒转化DH5α感受态细胞,所得阳性转化子经电泳分析并测序鉴定。将携带目的基因的穿梭质粒与辅助包装质粒共转染人胚肾细胞系HEK293细胞,反复冻融获取重组腺病毒,终点稀释法测定病毒滴度,重组腺病毒转染HEK293细胞并提取蛋白,采用免疫印迹法分析Ang-2蛋白的表达。结果PCR产物电泳分析可见大小约1.5 kp的特征性条带,基因序列显示测序结果与GenBank提供的Ang-2序列一致,经HEK293细胞包装后腺病毒滴度为1×10^(8.8)pfu/ml,免疫印迹法分析可见大小57 kD特征性条带,即重组腺病毒可成功表达Ang-2蛋白。结论 Ang-2基因重组腺病毒载体可成功构建。

Objective To construct the recombinant adenovirus vectors overexpressing angiopoietin-2（ Ang-2） in mice. Methods Mouse Ang-2 coding sequence was chemically synthesized and then was linked to the shuttle plasmid after amplification by PCR and identification. The recombinant plasmid carrying target gene was transformed into DH5α competent cells. The positive transformants were analyzed by electrophoresis and sequenced for identification. Shuttle plasmid carrying target gene and helper packaging plasmid were cotransfected into HEK293 cells. HEK293 cells were repeatedly frozen and thawed to obtain recombinant adenovirus. The titer of recombinant adenovirus was detected by end-point dilution method. The recombinant adenovirus was transfected into HEK293 cells,then Ang-2 protein was extracted and its expression was analyzed by Western blot. Results A characteristic band about 1. 5 kp was observed in the electrophoresis for PCR products. The gene sequencing showed that the sequence is identical to the Ang-2 gene sequence from Gene Bank. The titer of adenovirus was 1 × 10^8. 8 pfu/ml after packaging by HEK293 cells. Western blot analysis showed a characteristic band with the size of 57 kD,which indicated that the recombinant adenovirus could express Ang-2 protein. Conclusion The recombinant adenovirus vector of Ang-2 gene can be successfully constructed.