携带人生长抑素2型受体的腺病毒载体的构建和表达  

Construction and expression of recombinant adenovirus vector encoding human somatostatin receptor type 2

作　　者：徐彬[1] 徐浩[2] 李月琴[1] 曹忠健[1] 张欣[1] 周天鸿[1] 

机构地区：[1]暨南大学生物工程学系,广东广州510632 [2]暨南大学第一附属医院核医学科,广东广州510632

出　　处：《中国病理生理杂志》2006年第10期1997-2001,共5页Chinese Journal of Pathophysiology

基　　金：广东省重大科技计划项目资助(No.2003A3080501)

摘　　要：目的:构建表达SSTR2的重组腺病毒载体,用于胰腺癌的基因治疗。方法:构建重组腺病毒穿梭质粒pDC316-SSTR2,通过脂质体与腺病毒骨架质粒pBHG lox(delta)E1,3Cre共转染293细胞,经位点特异性重组获得重组腺病毒Ad-SSTR2。通过PCR鉴定Ad-SSTR2。经293细胞扩增,纯化制备高滴度病毒感染液。以病毒感染液感染人胰腺癌细胞capan-2,应用免疫印迹检测SSTR2蛋白的表达。结果:成功构建腺病毒穿梭质粒pDC316-SSTR2,与pBHG lox(delta)E1,3Cre共转染293细胞获得了重组腺病毒Ad-SSTR2,以Ad-SSTR2基因组DNA为模板同时扩增出了人SSTR2 cDNA基因片段和腺病毒骨架基因片段。Ad-SSTR2感染capan-2 48 h后,免疫印迹检测到SSTR2蛋白的表达。结论:表达人SSTR2的重组腺病毒载体构建成功,并在胰腺癌细胞中得到正确表达,为SSTR2基因治疗胰腺癌奠定了基础。AIM： To construct recombinant adenovirus vector carrying the gene of human somatostatin receptor type 2 （SSTR2） for gene therapy of pancreatic carcinoma. METHODS： SSTR2 cDNA was inserted into adenovirus shuttle plasmid pDC316, named pDC316- SSTR2. pDC316- SSTR2 was cotransfected with rescue plasmid pBHGlox （delta） El, 3Cre into 293 cells by liposome reagent. Ad - SSTR2 was generated by site - specific recombination and confirmed by PCR. Ad - SSTR2 was propagated in 293 cells and purified. The titer of viral stock was determined by end - point dilution assay. Western blotting was used to determine the expression of SSTR2 protein after human pancreatic carcinoma cell capan - 2 was infected with recombinant adenovirus. RESULTS： pDC316 -SSTR2 was successfully constructed. Recombinant adenovirus Ad -SSTR2 was acquired by pDC316 -SSTR2 and pBHGlox （delta） El, 3Cre cotransfected into 293 cells. Ad -SSTR2 was characterized by PCR. The virus titer was 6. 0 × 10^12 pfu/L. SSTR2 protein was detected after adenovirus infected capan -2 48 h with Western blotting. CONCLUSION： The recombinant adenovirus vector encoding human SSTR2 is successfully constructed and correctly expressed in pancreatic carcinoma cells. This investigation provides the basis for study of gene therapy of pancreatic carcinoma.

关 键 词：受体 生长抑素 腺病毒科 基因疗法 癌 

分 类 号：R363[医药卫生—病理学]