含新城疫病毒血凝素-神经氨酸酶基因重组腺病素的构建及鉴定  

Construction and identification of the recombinant adenovirus expressing hemagglutinin-neuramidinase of Newcastle disease virus

作　　者：陈漉[1] 金宁一[2] 李霄[2] 刘立明[2] 贾鹏[2] 刘妍[2] 高鹏[2,3] 陆蕴松[4] 李明[5] 迟宝荣[1] 

机构地区：[1]吉林大学第一医院消化科,长春130021 [2]军事医学科学院解放军基因工程重点实验室,长春130062 [3]吉林省人民医院血液科,长春130021 [4]吉林大学第三医院骨科,长春130033 [5]通化市中心医院,通化134001

出　　处：《中国免疫学杂志》2009年第2期132-135,共4页Chinese Journal of Immunology

基　　金：科技部国家高科技发展计划“863计划”(2007AA021004);吉林省科技发展计划项目(20060566)资助

摘　　要：目的：构建表达新城疫病毒血凝素-神经氨酸酶（Hemagglutinin—neuramidinase，FIN）基因的重组腺病毒，为进一步研究HN基因对消化道肿瘤的抑制作用及分子机制建立基础。方法：用BamHI和XbaⅠ双酶切质粒pVHN，将获得的HN基因片段连接入穿梭质粒pacAd5 CMV K-N pA，构建含HN基因的穿梭质粒pacAd5-HN。利用PacI单酶切对pacAd5-HN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：所构建重组病毒可有效表达HN基因，表达产物分子量约为63kD，重组病毒滴度为10^8～10610PFU／ml。结论：成功构建含HN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。Objective：To construet a recombinant adenovirus carrying HN gene so as to provide basis for further studying HN gene for molecular mechanisms by which to treat cancers in digestive organs. Methods： Reclaiming the recombinant plasmid pVHN was digested by double digestion of BamH Ⅰ and Xba Ⅰ with recovering hemagglutinin-neuramidinase （HN）inserts. HN fragments were ligated into the vector pacAd5 CMV K-N pA to produce the shuttle vector named as pacAd.5-CMV-K-N-pA-HN. Then the vector and pAD genome were linearized with Pac Ⅰ and transfected in AAV 293 cells by liposome systerm. The DNA containing HN encoding sequence of the recombinant adenovirus was identitied by plaque purification, RT-PCR and its expression was determined by Western blot. Results： HN gene in the recombinant adenovirus was expressed in the transfected AAV 293 cells and the molecular weight of the protein was about 65 kD. The titre of the recombinant adenovirus was up to 10^8 - 10^9 PFU/ml, sufficient for further in vivo and in vitro experiment. Conclusion： The recombinant adenovirus vector containing HN gene is successfully constructed and the target protein is fully expressed when transfected.

关 键 词：NDV HN基因 重组腺病毒 抗肿瘤 

分 类 号：R392.12[医药卫生—免疫学]