含Apoptin基因重组腺病毒的构建及鉴定  

Construction and identification of the recombinant adenovirus expressing Apoptin gene of chicken anemia virus

作　　者：陈漉[1] 金宁一[2] 李霄[2] 刘立明[2] 贾鹏[2] 刘妍[1] 高鹏[3] 陆蕴松[4] 迟宝荣[1] 

机构地区：[1]吉林大学第一临床医院消化内科,吉林省长春市130021 [2]中国人民解放军军事医学科学院全军基因工程重点实验室,吉林省长春市130062 [3]吉林省人民医院血液科,吉林省长春市130021 [4]吉林大学第三医院骨科,吉林省长春市130033

出　　处：《世界华人消化杂志》2008年第31期3505-3509,共5页World Chinese Journal of Digestology

基　　金：吉林省科技发展计划项目资助;No.20060566~~

摘　　要：目的:构建携凋亡素(Apoptin)基因重组腺病毒,为进一步研究Apoptin基因抗肿瘤作用分子机制建立基础.方法:利用BamHⅠ和SpeⅠ双酶切质粒pVAX1-Apoptin,获得Apoptin片段并连接入pacAd5 CMV K-N pA,构建含Apoptin基因的穿梭质粒pacAd5-Apoptin.利用PacⅠ单酶切对pacAd5-Apoptin和腺病毒基因组质粒(pAd5)进行线性化处理后应用脂质体介导法共转染AAV-293细胞,分别利用蚀斑纯化和RT-PCR、Western blot等方法对重组病毒进行筛选和鉴定,并测定所获得重组病毒的滴度.结果:所构建重组腺病毒中的Apoptin基因得到了正确转录,表达产物的相对分子质量约为13kDa,与CVA阳性对照一致;所获得重组腺病毒可有效表达Apoptin蛋白,该蛋白与CAV阳性血清具有反应原性,重组病毒滴度为1011PFU/L.结论:成功构建含Apoptin基因的重组腺病毒,所获得重组病毒的滴度可以满足体内外实验要求.AIM： To construct a recombinant adenovirus carrying Apoptin gene so as to provide a basis for further studying the molecular mechanism of Apoptin gene in inducing tumor cell apoptosis. METHODS： The plasmid pVAX1-Apoptin was digested by endonuclease BamH I and Spe I, and the obtained Apoptin segment was inserted into vector pacAd5 CMV K-N pA to construct a shuttle plasmid pacAd5-Apoptin. After Pac I digestion and linearized process, the plasmid pacAd5-Apoptin and pAD （genome plasmid） were co-transfected into AAV-293 cells by lipo- some mediation. The DNA containing Apoptin gene of the recombinant adenovirus was identi- fied by plaque purification, reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. The titer of the obtained adenovirus was also examined. RESULTS： The recombinant adenovirus expressed Apoptin gene and the molecular weight of the protein was about 13 kDa, which was con- sistent with the CVA-positive control. The protein of Apoptin could be effectively expressed in the recombinant adenovirus, and this protein had response to the CAV-positive serum. The titer of the recombinant virus was 10^11 PFU/L. CONCLUSION： The adenovirus containing Apoptin gene is successfully constructed, and the virus titer is able to meet the requirements of in vitro and in vivo experiments.

关 键 词：Apoptin基因 腺病毒载体 基因治疗 逆转 录-聚合酶链反应 免疫印迹法 

分 类 号：R346[医药卫生—基础医学]