HSV-1 UL30基因cDNA的克隆及筛选有效siRNA的融合载体构建  

Cloning of HSV-1 UL30 Gene cDNA and Construction of Fusion Expression Vector that Serves as Screening Effective siRNA

作　　者：何秋璟[1] 张春龙[2] 仁哲[3] 张美英[3] 朱钦昌[4] 刘秋英[3] 张佩琢 王一飞[3] 

机构地区：[1]广东医学院,广东东莞523808 [2]广东医学院衰老研究所,广东东莞523808 [3]暨南大学生物医药研究与开放基地,广东广州510632 [4]中科院广州生物医药与健康研究所,广东广州510663 [5]上海吉玛制药技术有限公司,上海201203

出　　处：《生物技术》2008年第3期1-4,共4页Biotechnology

基　　金：广东省重大攻关项目资助("昆布海藻抗病毒作用研究及其功能食品的开发";2005B50301017;2005A1090400)

摘　　要：目的:克隆HSV-1 UL30 cDNA并测序,构建pEGFP-N1-Fi融合表达载体,为靶向UL30基因的siRNA的设计和筛选奠定基础。方法:从感染HSV-1F株的病变VERO细胞提取总RNA,二次PCR扩增出UL30 cDNA并克隆至pEGFP-N1质粒,测序鉴定序列;将UL30 cDNA4个小片段亚克隆至pEGFP-N1形成pEGFP-N1-Fi融合表达质粒,转染VERO细胞,荧光显微镜观察融合蛋白表达情况。结果:成功克隆出UL30 cDNA,序列对比显示与基因库中的HSV-1F株UL30序列同源性为99.4%;成功构建pEGFP-N1-Fi融合表达载体并实现Fi-EGFP融合蛋白的表达。结论:成功克隆出UL30 cDNA,成功构建pEGFP-N1-Fi融合表达载体,为靶向UL30基因的siRNA的设计和筛选奠定基础。Objective：To clone and sequence HSV- 1 UL30 gene eDNA , and to construct the fusion expression vector pEGFP- N1 - Fi hying the foundation for designing and screening siRNA targeting UL30 gene. Method：Total RNA was abstracted from VERO cells infected with HSV - 1, UL30 eDNA was amplified via twice PCR and was cloned to the plasmid pEGFP - N1, sequence was identified via sequencing; the fusion expression vector pEGFP- N1 - Fi were constructed while 4 fragments of UL30 eDNA were subcloned to pEGFP- N1 and were transfected into VE- RO cells, expression of fusion protein was detected under fluorescence microscope. Result： UL30 eDNA was successfully cloned and shared the common sequence of 99.4% with the sequence in genebank, the fusion expression vector pEGFP- N1 - Fi was successfully constructed and the fusion protein Fi - EGFP was successfully expressed. Conclusion： UL30 eDNA was successfully cloned and the fusion expression vector pEGFP - N1 - Fi was successfully constructed laying the foundation for designing and screening siRNA targeting UL30 gene.

关 键 词：UL30基因/警纯疱疹病毒1型 分子克隆 EGFP 融合表达载体 

分 类 号：Q782[生物学—分子生物学]