携Apoptin和PEG3启动子双特异性重组腺病毒的构建及鉴定  

Construction and identification of a recombinant adenovirus carrying Apoptinand the PEG3 promoter

作　　者：兰添[1,2] 李一权 张诺娜 邢彬[2] 胡宁宁[2] 范园园[2] 陈爽[2] 姜爽[2] 于海玲[1] 李霄[2,3] 金宁一[2,3] 

机构地区：[1]延边大学医学院,吉林延吉133002 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国病原生物学杂志》2015年第11期966-970,共5页Journal of Pathogen Biology

摘　　要：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 use Apoptin and progression-elevated gene 3 （PEG3） to construct an oncolytic adenovirus, Ad-Apoptin-PEG3p-Ela, with specific ability to destroy cancer cells and to replicate in those cells, in order to provide a basis for the study of Apoptin＇s antitumor action. Methods Ela and PolyA were obtained from pMT-Ela and pIRES- neo and inserted into pKS-PEG3p. The plasmid was digested with XhoI and SpeI and then inserted into pAd-Apoptin to construct the shuttle plasmid pacAd-Apoptin-PEG3p-Ela. The plasmid pacAd-Apoptin-PEG3p-Ela and the genome plas mid pAd5 were co transfected into HEK293 cells to obtain the recombinant adenovirus Ad Apoptin-PEG3p-Ela. The re combinant adenovirus was identified using a reverse transcription polymerase chain reaction （RT-PCR）, Western blot ting, and electron microscopy. Its effects on A549 cells and 1.02 cells were assessed using an MTS assay and viral replica- tion was determined using a growth curve. Results The genome of the constructed adenovirus contained Apoptin, it had a normal morphology according to electron microscopy, and it inhibited A549 cell proliferation. Cell viability depen ded to a certain extent on the MOI of the recombinant adenovirus and the timing of administration. The inhibitory effects of the recombinant adenovirus peaked at 96 h and an MOI of 100. The rate of inhibition was 79.84±1.0%, which was significantly higher than that of the control adenovirus （P〈0.05）. The recombinant adenovirus had dual specificity in that it replicated in A549 cells while replicating little in L02 cells. Conclusion The recombinant adenovirus Ad Apop- tin-PEG3p-Ela was successfully constructed, and it had specific ability to inhibit cancer and replicate in cancer cells.

关 键 词：重组腺病毒 APOPTIN 进行性增量基因3 特异性 抗肿瘤 

分 类 号：R373[医药卫生—病原生物学]