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| 包装信号可以被Cre重组酶切除的辅助性腺病毒的构建及鉴定 | |
| 引用本文： | 董刚,俞卫锋,徐学武,吴飞翔,袁扬.包装信号可以被Cre重组酶切除的辅助性腺病毒的构建及鉴定[J].生物技术通讯,2010,21(4):461-465. |
| 作者姓名： | 董刚 俞卫锋 徐学武 吴飞翔 袁扬 |
| 作者单位： | 1. 解放军302医院,麻醉科,北京,100039;东方肝胆外科医院,麻醉科,上海,200438 2. 东方肝胆外科医院,麻醉科,上海,200438 3. 东方肝胆外科医院,麻醉科,上海,200438;解放军306医院,麻醉科,北京,100101 4. 第二军医大学,长海医院胸心外科实验室,上海,200433 |
| 摘 要： | 目的:为了探索构建第三代复制依赖性腺病毒载体系统的有效实验方法,利用细菌内重组原理,分别构建了第三代腺病毒生产体系中的辅助性腺病毒和表达Cre重组酶的真核表达载体,并分析了Cre切除辅助病毒包装信号的有效性。方法:通过PCR及酶处理法依次将2个同向的loxP位点及绿色荧光蛋白表达盒克隆至pShuttle穿梭载体质粒,按照AdEasy系统的标准实验方法产生和扩增辅助病毒;同时构建pcDNA3.1(+)-cre真核表达载体,并用该载体经脂质体法转染HEK293细胞,通过PCR和流式细胞术分析转染细胞内表达的Cre重组酶对随后感染的辅助病毒的包装信号的切除作用。结果:构建的辅助性腺病毒能够在HEK293细胞内扩增,其包装信号可以由细胞内表达的Cre重组酶有效切除。结论:构建了辅助性腺病毒和pcDNA3.1(+)-cre真核表达载体,为第三代腺病毒的生产奠定了实验基础。 |
| 关 键 词： | 第三代腺病毒载体 辅助性腺病毒 AdEasy系统 pcDNA3.1真核表达载体 Cre重组酶 |
Construction of Helper Adenoviruses and Analysis of Cre-Mediated Excision of the Packaging Signal |
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| DONG Gang,YU Wei-Feng,XU Xue-Wu,WU Fei-Xiang,YUAN Yang.Construction of Helper Adenoviruses and Analysis of Cre-Mediated Excision of the Packaging Signal[J].Letters in Biotechnology,2010,21(4):461-465. | |
| Authors: | DONG Gang YU Wei-Feng XU Xue-Wu WU Fei-Xiang YUAN Yang |
| Institution: | DONG Gang1,2,YU Wei-Feng2,XU Xue-Wu2,3,WU Fei-Xiang2,YUAN Yang4 1. Department of Anesthesiology,302nd Hospital of PLA,Beijing 100039,2. Department of Anesthesiology,Eastern Hepatobiliary Surgery Hospital,Shanghai 200438,3. Department of Anesthesiology,306th Hospital of PLA,Beijing 100101,4. Laboratory of Cardio-Thoracic Surgery,Changhai Hospital,Second Military Medical University,Shanghai 200433,China |
| Abstract: | Objective: To investigate the approach to construct the third-generation helper-dependent adenovirus vector system, a helper virus in the helper-dependent adenovirus production system and a mammalian expression vector expressing Cre recombinase were constructed, respectively, then the effectiveness of excision of Cre-mediated packaging signal in helper viruses was analyzed. Methods: Two parallel loxP sites and EGFP gene were cloned into pShuttle one by one by overlapping PCR or cleavage and ligation followe... |
| Keywords: | the third-generation adenovirus vector helper adenoviruses AdEasy system pcDNA3 1 mammalian expression vector Cre recombinase |
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