| 利用Gateway克隆技术构建人Hiwi腺病毒载体 |
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| 引用本文： | 马宁,董晓燕,姜艳芳,刘蒙蒙,刘子玲.利用Gateway克隆技术构建人Hiwi腺病毒载体[J].吉林大学学报(医学版),2014,0(4):898-903. |
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| 作者姓名： | 马宁 董晓燕 姜艳芳 刘蒙蒙 刘子玲 |
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| 作者单位： | 1．吉林大学第一医院血液肿瘤科，吉林 长春 130021；2. 吉林大学第一医院风湿免疫科，吉林 长春 130021；3. 河南省人民医院血液病研究所，河南 郑州 450000；4. 吉林大学第一医院二部中心研究室，吉林 长春 130031 |
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| 基金项目： | 吉林省科技厅科研基金资助课题(项目编号：200705181) |
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| 摘 要： | 目的：获得人的全长Hiwi编码区基因，构建携带绿色荧光蛋白（GFP）的人Hiwi腺病毒载体,为进一步研究Hiwi诱导白血病干细胞分化和凋亡的作用和机制奠定基础。方法：运用重叠延伸PCR方法扩增Hiwi编码区基因全长；利用Gateway克隆技术将Hiwi编码区基因全长插入带有GFP的载体Flag-IRES -hrGFP中构建成为pDown-Hiwi-3×flag-IRES-hrGFP，再将克隆载体pDown-Hiwi-3×flag-IRES-hrGFP与表达载体pAV.Des1d进行同源重组反应，得到重组腺病毒载体pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP。经PCR筛选阳性克隆提取重组腺病毒质粒，包装成重组Ad-Hiwi-3×flag-IRES-hrGFP腺病毒。结果：人Hiwi全长编码区基因克隆成功，经酶切及基因测序鉴定证实重组Ad-Hiwi-3×flag-IRES-hrGFP腺病毒质粒构建成功。采用Lipofectamine法将Ad-Hiwi-3×flag-IRES-hrGFP转染入HEK293A细胞中，在荧光显微镜下，可以观察到转染细胞中的绿色荧光。结论：成功构建重组Ad- Hiwi-3×flag-IRES-hrGFP腺病毒质粒，其可在HEK293A细胞内表达。
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| 关 键 词： | Hiwi基因 腺病毒 重叠延伸聚合酶链反应 Gateway克隆技术 |
| 收稿时间： | 2014-01-10 |
Construction of recombinant adenovirus vector pAV.Ex1d-Hiwi using Gateway technology |
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| MA Ning,DONG Xiao-yan,JIANG Yan-fang,LIU Meng-meng,LIUZi-ling.Construction of recombinant adenovirus vector pAV.Ex1d-Hiwi using Gateway technology[J].Journal of Jilin University: Med Ed,2014,0(4):898-903. |
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| Authors: | MA Ning DONG Xiao-yan JIANG Yan-fang LIU Meng-meng LIUZi-ling |
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| Institution: | 1.Department of Hematologic Neoplasms,First Affiliated Hospital,Jilin University,Changchun 130021,China；2. Department of Rheumatology,First Hospital,Jilin University,Changchun 130021,China；3.Institute Hematology, of Henan Provincial People’s Hospital,Zhengzhou 450000,China；4. Departmentof Central Laboratory,Second Part of First Hospital,Jilin University,Changchun 130031,China |
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| Abstract: | Objective To obtain the Hiwi gene encoding full-length and construct human Hiwi adenoviral vectors carrying green luorescence protein(GFP),and to establish foundation for a further study on Hiwi function and mechanism of inducing leukemia stem cell differentiation and apoptosis.Methods All coding areas of human Hiwi gene full length were amplified using method of overlapping extension PCR technology,and the full length coding aeras were inserted into the vector of Flag-IRES-hrGFP carrying GFP with Gateway technology to construct pDownHiwi-3×flag-IRES-hrGFP.The cloning vector pDown-Hiwi-3×flag-IRES-hrGFP and expression vector pAV.Des1d were used for homologous recombination reaction to obtain recombinant adenovirus vector pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP.The positive clones were selected by PCR to extract the recombinant adenovirus plasmid and to pack into recombinant Ad-Hiwi-3×flag-IRES-hrGFP adenovirus.Results The human recombinant Hiwi was successfully cloned and the recombinant adenovirus vector pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP was found to be successfully constructed via restriction enzyme digestion and sequencing methods.The adenovirus vector pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP was transfected into the HEK293A cells using lipofectamine mediated gene transfection method.Under fluorescence microscope,the transfected cells with green fluorescence could be observed.Conclusion The expression plasmid of adenovirus vector pAV.Ex1d-Hiwi-3×flag-IRES-hrGFP is successfully constructed and it can express in HEK293A cells. |
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| Keywords: | Hiwi gene adenovirus overlapping extension polymerase chain reaction Gateway technology |
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