携带人血管生成素-1腺病毒表达载体Padeasy-1/Ang-1的构建、鉴定和滴度测定  

Construction,identification and titer detection of adenovirus expression vector Padeasy-1/ Ang-1 carrying human angiopoietin-1

作　　者：殷骏[1] 梁志清[1] 王丹[1] 

机构地区：[1]第三军医大学西南医院妇产科,重庆400038

出　　处：《重庆医学》2009年第10期1190-1192,共3页Chongqing medicine

基　　金：国家自然科学基金资助项目(30600673);"十一五"全军科技计划青年基金项目(06Q054)。

摘　　要：目的克隆Ang-1基因,构建Ang-1/Pshuttle/Padeasy-1腺病毒表达载体,为进一步研究Ang-1防治卵巢癌病变的血管异质性提供理论基础。方法提取流产胎儿肝脏组织总RNA,RT-PCR扩增出Ang-1基因片段,并将其克隆到Pshuttle载体中进行序列分析,经PacⅠ线性化、连接到腺病毒表达载体Padeasy-1中,将Padeasy-1包装进脂质体、转染入人胚肾293细胞,测定病毒生物活性滴度。结果从流产胎儿肝脏组织总RNA中,扩增出1.4kb的cDNA片段,成功构建了Padeasy-1/Pshuttle/Ang-1表达载体。结论从流产胎儿肝脏组织中成功克隆Ang-1基因,成功构建Padeasy-1/Pshuttle/Ang-1表达载体,Padeasy-1/Pshuttle/Ang-1表达载体的滴度为1×1011PFU/mL。Objective To clone angiopoietin-1 genes and construct Ang-1/Pshuttle/Pageasy-1 adenovirus expression vector, so as to make theory base for the further research on the Ang-1 prevention and cure blood vessel heterogenous of ovarian caner pathological changes. Methods Extracting mRNA from abortion embryo liver,amplified Ang-1 gene by RT-PCR and cloned into Pshuttle vector and sequenced,linearized by Pac Ⅰ and connected to adenovirus expression vector Padeasy-1, packaging Padeasy into liposome,transfect to 293 cells, determination of virus biological activity titer. Results From abortion embryo liver total RNA, RT- PCR amplified 1. 4kb cDNA fragment, successfully constructing Padeasy-1/Pshuttle/Ang-1 express vector. Conclusion Cloned Ang-1 gene and successfully constructing Padeasy-1/Pshuttle/Ang-1 express vector,it＇s titer is 1 × 10^11PFU/mL.

关 键 词：血管生成素-1 腺病毒载体 基因克隆 

分 类 号：R341[医药卫生—基础医学]