EEF1A2嵌合型重组腺病毒载体的构建及鉴定

Construction and identification of human chimeric recombinant adenovirus with EEF1A2 gene

目的:克隆人类真核翻译延长因子1A2(EEF1A2)基因全长,构建其重组腺病毒载体。方法:应用RT-PCR方法从人胰腺癌组织中扩增人EEF1A2基因全长,经T-A克隆后,亚克隆到腺病毒穿梭质粒pDC316,构建穿梭质粒pDC316-EEF1A2。利用同源重组的方法将腺病毒骨架质粒pBHG35和穿梭质粒pDC316-EEF1A2共转染293细胞,获得腺病毒重组质粒Ad5/F35-EEF1A2,经过在293细胞中包装和扩增之后,获得高滴度的腺病毒重组质粒Ad5/F35-EEF1A2。PCR、WesternBlot检测EEF1A2基因的表达。结果:用RT-PCR方法,从人胰腺癌组织中扩增出1400bp的cDNA片段,经测序证实为人EEF1A2基因。构建及包装出高滴度的重组腺病毒载体Ad5/F35-EEF1A2。结论:成功地克隆了人EEF1A2基因全长,并构建其表达的重组腺病毒载体,为进一步研究人EEF1A2基因在相关疾病中的作用奠定了基础。

Objective To clone EEF1A2 full-length cDNA and construct its recombinant adenovirus vector. Methods The EEF1A2 gene was amplified by reverse transcription polymerase chain reaction （RT-PCR） from human pancreatic cancer tissues and then cloned into adenovirus shuttle plasmid pDC316 to form pDC316-EEF1A2 after T-A cloning. The pDC316-EEF1A2 plasmid and the framework plasmid pBHG35 were cotransfected into HEK293 cells to construct adenovirus recombinant plasmid Ad5/F35-EEF1A2 and a high-titer Ad5/F35-EEF1A2 was generated after packing and amplification in the 293 cell. The target gene was detected by polymerase chain reaction （PCR） and Western Blot. Results A 1 400 bp cDNA was amplified by RT-PCR from human pancreatic cancer tissues, which was identified by sequence analysis as the EEF1A2 gene. A high-titer Ad5/F35-EEF1A2 vector was constructed and packed. Conclusion EEF1A2 gene has been successfully cloned and its recombinant adenovirus vector has been constructed, which lays a foundation in further research on the role of EEF1A2 gene in some related disorders.