EBV即刻早期基因BZLF1腺病毒表达载体的构建及初步应用

Construction and initial application of recombinant adenoviral expressed vector carrying EBV immediately early gene BZLF1

目的构建EBV即刻早期基因BZLF1的重组腺病毒表达载体,探讨其表达诱导潜伏状态EBV进入裂解期增殖的作用。方法采用AdEasy系统构建携带BZLF1的重组腺病毒载体pAd- BZ,脂质体法转染人胚肾293细胞,包装产生重组腺病毒vAd-BZ。vAd-BZ感染EBV阳性细胞NEC后,采用RT-PCR、Western blot、流式细胞术和MTT等方法检测目的基因表达,以及目的基因表达对EBV阳性细胞的影响。结果PCR、序列测定以及限制性酶切证实BZLF1基因正确插入穿梭质粒,并与病毒骨架质粒pAdEasy-1重组,重组腺病毒表达载体构建成功。经293细胞包装获得具有稳定感染性的重组腺病毒vAd-BZ,感染重组腺病毒的NEC靶细胞可以检测到BZLF1基因的表达,进而诱导EBV从潜伏期进入裂解期。结论重组腺病毒vAd-BZ可有效感染EBV阳性细胞NEC,诱导潜伏状态的EBV活化,进而特异性杀伤EBV阳性肿瘤细胞。

Objective To construct recombinant adenoviral express vector carrying EBV immediately early gene BZLF1 and to investigate its function of inducing latent EBV into lyric cycle in vitro. Methods The recombinant adenovirus vector carrying EBV immediately early gene BZLF1 was constructed with AdEasy system and then transfected 293 cells to create recombinant adenovirus vAd-BZ. EBV positive NEC cells were infected with recombinant adenoviruses. The expression of target germ and its effects on EBV positive cells were tested by RT-PCR, Western blot, FACS and MTT in vitro. Results The recombinant adenovirus vector was constructed successfully. The recombinant adenovims vAd-BZ, which has stable infectivity, was packed in 293 cells. BZLF1 can be detected in EBV positive cells NEC infected by recombinant adenovirus and EBV was induced to enter lytic form after infection. Conclusion The recombinant adermviruses vAd-BZ can effectly infect EBV positive NEC cells, induce a switch of EBV from latency to lyric cycle and kill EBV positive tumor cells specifically, which is potential for gene therapy of EBV associated tumor.