携带Apoptin及hTERT启动子的双重特异性重组腺病毒的构建及鉴定  

Construction and identification of a recombinant adenvirus carrying apoptin and the hTERT promoter

作　　者：艾迪[1] 李文杰[1,2] 李一权 陈爽 朱羿龙[1] 崔英丽 李敏[1] 崔传信 尹逊哲[1] 李善智[1] 马艺珍 李霄 金宁一 AI Di1 , LI Wen-jie1,2 , LI Yi-quan1,3 , CHEN Shuang1,3,4 , ZHU Yi-long1 , CUI Ying-li1 , LI Min1 , CUI Chuang-xin1 ,YIN Xun-zhe1 ,LI Shan-zhi1 ,MA Yi-zhen1 ,LI Xiao1 ,JIN Ning-yi1(1. Institute of Military Veterinary Medicine ,Academy of Military Medical Sciences, Changchun 130122,China; 2. Animal Science and Technology College of Guangxi University, Nanning 530004, China;3. Medical College of Yanbian University ,Yanji, Jilin 133002, China; 4. Inspection College of Jilin Medical University ,Jilin City ,Jilin 132013,Chin)

机构地区：[1]军事医学科学院军事兽医研究所,吉林长春130122 [2]广西大学动物科学技术学院,广西南宁530004 [3]延边大学医学院,吉林延吉133002 [4]吉林医药学院检验学院,吉林吉林132013

出　　处：《中国兽医学报》2018年第5期937-941,955,共6页Chinese Journal of Veterinary Science

基　　金：吉林省产业技术创新战略联盟项目(20140309006YY);吉林省重大科技攻关项目(20150201002YY)

摘　　要：从质粒pMT-E1a和pIRES-neo中获得目的基因E1a和PolyA并连接入质粒pKS-hTERTp，用Xh0I和SpeI双酶切后连接入线性化的pAd—Apoptin，获得穿梭载体pAd—apoptin-PolyA—hTERTp—Ela。穿梭载体pAd—Apoptin-PolyA—hTERTp—Ela和腺病毒骨架质粒（pAd5）共转染HEK293细胞，获得重组腺病毒Ad—Apoptin-hTERTp—Ela，采用RT—PCR和Westernblot对重组腺病毒进行鉴定，MTs法检测病毒对A549细胞的抑制作用；绘制重组腺病毒生长曲线，监测重组腺病毒在细胞内的复制能力。结果显示：构建出的重组腺病毒中包含有了目的基因Apoptin和E1a，并且目的基因在细胞中正确的表达；MTs结果显示重组腺病毒对A549具有抑制作用，且抑制作用具有一定的时效及剂效关系；重组腺病毒具有在A549细胞中正常复制的能力。结果表明：成功的构建出了具有特异性杀伤和特异性复制能力的双重特异性重组腺病毒Ad—Apoptin-hTERTp—E1a。The target genes Ela and PolyA were obtained from plasmids pMT-Ela and pIRES-neo and ligated into plasmid pKS-hTERTp. After digested with Xho Ⅰ and Spe Ⅰ ,ligated into linearized pAd-Apoptin to obtain shuttle vector pAd-Apoptin-PolyA-hTERTp-Ela. The recombinant adenovirus Ad-Apoptin-hTERTp-Ela was co-transfected into HEK293 cells with shuttle vector pad- Apoptin-PolyA-hTERTp-Ela and adenovirus backbone plasmid pAd5. The recombinant adenovirus was identified by RT-PCR and Western blot MTS assay was used to detect the inhibitory effect of the virus on A549 ceils. The growth curve of recombinant adenovirus was drawn to detect the replication of the recombinant adenovirus in the cell. The recombinant adenovirus containing the target genes Apoptin and Ela, can be correctly expressed in cells ; MTS results showed that the recombinant adenovirus inhibitied A549,and the inhibition has a certain aging and efficacy relationship;recombinant adenovirus normaly replicated in tumor cells A549. The recombinant adenovirus Ad-Apoptin-hTERTp-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 凋亡素 人端粒酶末端转移酶 特异性 抗肿瘤 

分 类 号：S852.65[农业科学—基础兽医学] S855[农业科学—兽医学]