负载肝素酶复制缺陷型腺病毒的包装及鉴定  

Package and identification of replication-deficient recombinant adenovirus expression vector of human heparanase

作　　者：陈陵[1] 蔡永国[1] 郑兴春[1] 杨仕明[1] 房殿春[1] 罗元辉[1] 王东旭[1] 

机构地区：[1]第三军医大学西南医院全军消化中心,重庆400038

出　　处：《解放军医学杂志》2006年第1期38-41,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(30200123)

摘　　要：目的包装负载人肝素酶(hpa)全长cDNA的复制缺陷型腺病毒载体(Adhpa)。方法将肝素酶正义腺病毒穿梭质粒pDC315hpa与腺病毒包装质粒pBHGloxDeltaE1,3Cre共转染人胚肾293细胞(HEK293),包装Adhpa,并大量扩增,收集纯化。对纯化后的Adhpa进行滴度测定、感染性鉴定、电镜鉴定及双引物PCR鉴定。结果包装成功的腺病毒载体具有良好的感染性,纯化浓缩后的病毒滴度可达5×1010pfu/ml。电镜证实病毒浓缩液中存在病毒颗粒,并可见病毒在HEK293细胞中复制。PCR双引物鉴定Adhpa可扩增出Ad及hpa特异片段,而对照则不能扩增出hpa片段。结论成功包装了负盘。Objective To construct a replication-deficient recombinant adenovirus expression vector of human heparanase （hpa）. Methohs The hpa gene was cloned at the downstream of CMV promoter of the adenoviral shuttle plasmid pDC315 in sense direction, and the resultant recombinant plasmid pDC315-hpa was transfected into HEK293 cell together with plasmid pBHGlox （deltaE1,3） containing adenoviral genome, then the replication-deficient recombinant adenovirus expression vector of hpa （Ad-hpa） was obtained, and it was identified by infection test, electronic microscope observation and PCR amplification. Results After purification and concintration, the titer of Ad-hpa reached 5 × 10^10 pfu/ml. Virus particles could be found in virus concintration solution, and replication of virus was observed in HEK293 cells was observed under transmission electron microscope. Both adenovirus and hpa special fragment could be amplified from Ad-hpa by PCR, whereas hpa special fragment could not be amplified from the control. Conclusion The replication-deficient recombinant adenovirus expression vector of hpa was constructed successfully. This study established a foundation for further study on hpa vaccines and gene therapy for carcinoma.

关 键 词：肝素酶 腺病毒科 复制缺陷型腺病毒载体 转染 

分 类 号：Q782[生物学—分子生物学]