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目的利用凋亡素基因(Apoptin)和进行性增量基因3(progression-elevated genes 3,PEG3)构建具有特异性杀伤和特异性复制功能的双特异性溶瘤腺病毒Ad-Apoptin-PEG3p-E1a,为研究Ad-Apoptin-PEG3p-E1a的抗肿瘤作用奠定基础。方法从pMT-E1a和pIRES-neo获得目的基因E1a和PolyA并连接入pKS-PEG3p,用XhoⅠ和SpeⅠ双酶切后连接入pAd-Apoptin,获得穿梭载体pacAd-Apoptin-PEG3p-E1a。用pacAd-Apoptin-PEG3p-E1a和腺病毒骨架质粒(pAd5)共转染HEK293细胞,获得重组腺病毒Ad-Apoptin-PEG3p-E1a,利用RT-PCR、Western blot和电镜对重组病毒进行鉴定,采用MTS检测病毒对A549细胞的抑制效果及其对L02细胞的影响;通过绘制一步生长曲线,检测重组毒在细胞内的复制情况。结果构建的重组病毒基因组中含有Apoptin目的基因,电镜下具有正常形态。MTS检测重组腺病毒对A549细胞的抑制作用具有时效和量效关系(P<0.05),抑制作用在100MOI、96h时达峰值为(79.84±1.0)%,对正常细胞L02无显著影响,该双特异性重组腺病毒能够在A549肿瘤细胞内复制,在L02细胞内几乎无复制能力。结论成功包装重组腺病毒Ad-Apoptin-PEG3p-E1a,且该病毒具有肿瘤特异性杀伤和肿瘤特异性复制功能。
OBJECTIVE: To construct a new type of oncolytic adenovirus Ad-Apoptin-PEG3p-E1a with specific killing and specific replication using Apoptin and progression-elevated genes 3 (PEG3) , Which lays the foundation for studying the anti-tumor effect of Ad-Apoptin-PEG3p-E1a. Methods The target genes E1a and PolyA were obtained from pMT-E1a and pIRES-neo and ligated into pKS-PEG3p. After digested with XhoⅠand SpeⅠ, the recombinant plasmid was ligated into pAd-Apoptin to obtain the shuttle vector pacAd-Apoptin-PEG3p-E1a. The recombinant adenovirus Ad-Apoptin-PEG3p-E1a was co-transfected with pacAd-Apoptin-PEG3p-E1a and adenovirus backbone plasmid (pAd5), and the recombinant virus was identified by RT-PCR, Western blot and electron microscopy. MTS detection of virus A549 cells and its effect on L02 cells inhibitory effect; by drawing a one-step growth curve to detect recombinant virus replication in the cell. Results The constructed recombinant virus genome contained the target gene of Apoptin and had normal morphology under electron microscope. The inhibitory effect of MTS on A549 cells was time-and dose-dependent (P <0.05). The inhibitory effect of MTS on the proliferation of A549 cells at 100 MOI peaked at (79.84 ± 1.0)% at 96 h and no significant effect on L02 cells Specific recombinant adenovirus can replicate in A549 tumor cells with almost no replication ability in L02 cells. Conclusion The recombinant adenovirus Ad-Apoptin-PEG3p-E1a was successfully packaged and the virus has tumor-specific killing and tumor-specific replication.