自杀基因TK腺相关病毒载体的构建及其抑制肿瘤细胞的功能研究

Construction of pAAV-TK and identification of its function of anticancer

目的探讨腺相关病毒（rAAV）-单纯疱疹病毒胸苷激酶基因（herpes simplex virus thymidinekinase,HSV-TK）（简称rAAV-TK）载体的构建及其生物学效应的初步鉴定。方法用PCR扩增TK序列至pAAV-MCS载体,构建pAAV-TK;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,对病毒颗粒进行鉴定;用MB49-PSA细胞对rAAV-TK生物学效应进行初步鉴定。结果成功构建pAAV-TK载体,并经酶切、PCR及测序验证其准确性;rAAV病毒颗粒滴度达2×1010 v.p/mL,浓缩后可达到2×1011~2×1012 v.p/mL;rAAV-TK可以分泌TK基因,加入药物更昔洛韦（Ganciclovir,GCV）和吉地他滨（Gemcitabine,GVC）,该基因能有效诱导膀胱肿瘤细胞MB49-PSA凋亡。结论成功构建rAAV-TK腺相关病毒,体外实验表明rAAV-TK/GVC/GCV系统能高效诱导膀胱肿瘤细胞的凋亡。

【Objective】 To construct pAAV-TK（herpes simplex virus thymidine kinase） and to identify its function.【Methods】 TK fragment from pUC57-TK obtained by PCR and then cloned into vector pAAV-MCS to construct pAAV-TK.Viral particle of purified rAAV was assayed by AVSachTM ELISA.The function of rAAV-TK was identified via MB49-PSA cell.【Results】 pAAV-TK was constructed and tested by sequence identification and enzyme digestion.We obtained high quality of rAAV after dissociating and purifying.The viral particles titre of rAAV was 2×1011~2×1012 v.p/mL.Together with ganciclovir and gemcitabine,rAAV-TK had effective function of TK by inducing cell apoptosis of MB49-PSA cell.【Conclusion】 rAAV-TK is successfully constructed,it can inhibit bladder cancer cell induced angiogenesis.