肿瘤血管内皮抑素tumstatin腺相关病毒载体的构建及其功能的初步鉴定

Construction of pAAV-Tum and identification of its function

目的:探讨腺相关病毒(rAAV)-肿瘤内皮抑素(tumstatin)(简称rAAV-Tum)载体的构建及其生物学效应的初步鉴定。方法:用PCR扩增tumstatin序列至pAAV-MCS载体,构建pAAV-Tum;采用AAV-Helper Free System包装系统、"氯仿-PEG/NaCl沉淀-氯仿抽提"及冰乙醇沉淀法进行AAV的包装、纯化和浓缩;用人脐静脉内皮细胞(HUVEC)对rAAV-Tum生物学效应进行初步鉴定。结果:成功构建pAAV-Tum载体,并经酶切、PCR及测序证实;rAAV病毒颗粒滴度达2×1010v.p/mL,浓缩后可达到2×1011～12v.p/mL;rAAV-Tum可以分泌内皮抑素,诱导HUVEC凋亡。结论:成功构建rAAV-Tum腺相关病毒,体外实验表明该病毒能高效诱导内皮细胞的凋亡。

Objective To construct pAAV-Tum and to identify its function. Methods Tumstatin fragment from pUC57-Tum attained by PCR and then cloned into vector pAAV-MCS to construct pAAV-Tum. Viral particle of purified rAAV was assayed by AVSachTM ELISA. Identify the function of rAAV-Tum via HUVEC cell. Results pAAV-Tum was constructed and tested by sequence indentifieation and enzyme digestion. We obtained high quality of rAAV after dissociating and purifying. The viral particles titre of rAAV were 2 × 10 11 - 12 v.p/mL, rAAV-Tum has function of tumstatin gene by inducing cell apoptosis of HUVEC cell. Conclusion We successfully constructed rAAV-Tum, it can inhibit tumor induced angiogenesis.