靶向性治疗EBV阳性淋巴瘤的腺病毒载体的构建及初步鉴定

Construction and identification of the recombinant adenovirus vector targeting EBV-positive lymphoma

目的:构建靶向EBV阳性淋巴瘤且携带野生型P53基因的重组腺病毒。方法:构建重组质粒pDC311.orip-CMV.P53.IRES-EGFP,后将该质粒与腺病毒骨架质粒PBHGE3共转染293细胞,包装成rAd5.orip-CMV.P53。PCR方法对病毒鉴定后,TCID50法测定其滴度,同时用该病毒分别感染EBV阳性Raji细胞和EBV阴性Jurket细胞,以观察病毒的特异性。结果:成功地构建了能表达P53靶向EBV阳性淋巴瘤的重组腺病毒,病毒的滴度为3.6×1012pfu/L。体外细胞实验显示该病毒具有一定特异性。结论:成功构建rAd5.orip-CMV.P53载体,为靶向治疗EBV阳性淋巴瘤奠定基础。

Objective：To construct the recombinant adenovirus vector which can target EB virus positive lymphoma and express wild-type P53 gene. Methods：The rAd5.orip-CMV.P53 was generated by contransfection of 293 cells with pDC311.orip-CMV.P53. IRES-EGFP and the adenovirual packaging plasmid PBHGE3. The construction of virus was identified by PCR method, and the titer was measured by TCID50. To observe the specificity of the recombinant adenovirus, EBV-positive raji ceils and EBV-negitive jurket cells were infected using rAd5.orip-CMV.P53 respectively. Results ：The recombinant adenovirus targeting EBV-postive lymphoma and expressing P53 gene was successfully constructed. The results in vitro showed the recombinant adenovirus has specificity to EBV- positive lymphoma cells. Conclusion：rAd.orip-CMV.P53 may be used for targeting gene therapy of EBV-positive lymphoma.