携带hTERT I540-PTD表达框重组复制缺陷型腺病毒的构建与鉴定  

Construction and identification of recombinant replication-defective adenoviruses harbouring expression cassettes of I540-PTD and I540-PTD4

作　　者：郭晓彤[1] 黎静[1] 薛妍[2] 宣敏[1] 李立文[3] 

机构地区：[1]广州军区广州总医院肿瘤科,广东广州510010 [2]第四军医大学西京医院肿瘤科,陕西西安710033 [3]西北大学生命科学学院,陕西西安710069

出　　处：《第四军医大学学报》2009年第9期796-799,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30600750);陕西省自然科学基金[2006K09-G9(5)]

摘　　要：目的:构建携带人端粒酶逆转录酶亚基(hTERT)I540-PTD,I540-PTD4融合基因表达框的重组复制缺陷型腺病毒.方法:将pDC315-I540-PTD,pDC315-I540-PTD4与腺病毒基因组质粒pBHGloxdeltaE13Cre共转染HEK293细胞,出现典型的细胞病变反应后收获细胞,反复冻融法制备病毒粗提液,经扩增和纯化获得重组复制缺陷型腺病毒Ad-I540-PTD和Ad-I540-PTD4.使用终点稀释实验和空斑形成实验测定纯化后病毒滴度.采用聚合酶链式反应(PCR)对Ad-I540-PTD,Ad-I540-PTD4进行鉴定.使用病毒感染人骨肉瘤U2OS细胞后,进行免疫组化定性并观察I540-PTD,I540-PTD4的表达情况.结果:共转染10d后,HEK293细胞出现典型的细胞病变反应.经扩增、纯化后,终点稀释法和孔板形成法测定Ad-I540-PTD滴度分别为(8.9×1012),(6.3×1012)pfu/L;Ad-I540-PTD4滴度分别为(5.8×1012),(5.4×1012)pfu/L.以纯化的病毒为模板,使用自行设计的鉴定引物进行PCR均获得167bp目标片段.抗组胺酸标签抗体对病毒感染后的U2OS细胞免疫组化分析发现Ad-I540-PTD,Ad-I540-PTD4转染的U2OS细胞均呈强染,表明Ad-I540-PTD,Ad-I540-PTD4中携带的I540-PTD,I540-PTD4真核表达读框可在人类细胞内高效表达.结论:成功构建可高效表达I540-PTD,hTERTI540-PTD4的重组复制缺陷型重组腺病毒Ad-I540-PTD,Ad-I540-PTD4,为其作为基因疫苗诱发针对hTERT I540表位的抗瘤免疫奠定了基础.AIM： To construct the recombinant replicationdefective adenoviruses harbouring the expression cassettes of 1540- PTD and I540-PTD4 fusion genes. METHODS： Recombinant adenovirus was constructed by co-transfecting HEK293 cells with pDC315-I540-PTD and pDC315-I540-PTD4 with genomic plasmid pBHGloxdeltaE13Cre respectively. The infectious titers of Ad-I540-PTD and Ad-I540-PTD4 were tittered by end-point dilution assay and plaque assay. After infected with the recombinant viruses, the expression of I540-PTD and I540-PTD4 fusion proteins was analyzed by immunohistochemical staining. RESULTS： Ten days after co-transfecting HEK293 cells with pDC315-I540-PTD and pDC315-I540-PTD4 with genomic plasmid pBHGloxdeltaE13Cre respectively, typical cytopathic effects were seen. The lysates containing recombinant replication-defective adenoviruses Ad-1540-PTD and Ad-I540-PTD4 were obtained by three consecutive free-thaw cycles after harvesting the HEK293 cells. The infectious titers of Ad-I540-PTD and Ad-I540-PTD4 were （ 8.9 × 1012）and （6.3 × 10^12 ） pfu/L, （5.8 × 1012 ） and （5.4 × 10^12 ） pfu/L determined respectively by end-point dilution assay and by plaque assay. Ad-I540-PTD and Ad-I540-PTD4 were confirmed by amplifying the 167 bp products by PCR with specific primers using recombinant adenoviruses DNA as templates. Immunohistochemical analysis using anti-His tag monoclonal antibody demonstrated that the U20S infected with Ad-I540-PTD and Ad-I540- PTD4 was both strongly positively stained. CONCLUSION： We have successfully designed and constructed the recombinant replica- tion-defective adenoviruses Ad-I540-PTD and Ad-I540-PTD4 harbouring I540-PTD and I540-PTD4 expression cassette respectively.

关 键 词：癌症疫苗 端粒酶逆转录酶 肿瘤 免疫疗法 

分 类 号：R730[医药卫生—肿瘤]