[1]梁 金,沈 宁,徐 康,等.cnksr2基因干扰腺病毒的构建及鉴定[J].南京师大学报(自然科学版),2013,36(03):103-107.
　Liang Jin,Shen Ning,Xu Kang,et al.Construction and Identification of cnksr2 Gene Interference Adenovirus[J].Journal of Nanjing Normal University(Natural Science Edition),2013,36(03):103-107.
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cnksr2基因干扰腺病毒的构建及鉴定()
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《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第36卷
期数:
2013年03期
页码:
103-107
栏目:
生命科学
出版日期:
2013-09-30

文章信息/Info

Title:
Construction and Identification of cnksr2 Gene Interference Adenovirus
作者:
梁 金1; 沈 宁1; 徐 康2; 李朝军1; 薛 斌1
(1.南京大学医学院,江苏省医学分子技术重点实验室,江苏 南京 210093) (2.南京师范大学生命科学学院,江苏省分子医学生物技术重点实验室,江苏 南京 210023)
Author(s):
Liang Jin1; Shen Ning1; Xu Kang2; Li Chaojun1; Xue Bin1
(1.Medical School of Nanjing University,Jiangsu Key Laboratory for Molecular Medicine,Nanjing 210093,China) (2.School of Life Sciences,Nanjing Normal University,Jiangsu Key Laboratory for Molecular and Medical Biotechnology,Nanjing 210023,China)
关键词:
cnksr2基因; 干扰; 腺病毒
Keywords:
cnksr2; interference; adenovirus
分类号:
Q291
摘要:
构建小鼠cnksr2基因干扰腺病毒质粒载体并加以鉴定.根据小鼠cnksr2基因序列设计cnksr2干扰序列引物,定向克隆至穿梭载体pshuttle-H1的Bgl Ⅱ和Hind Ⅲ位点,Pme Ⅰ酶切线性化的pShuttle-H1-Sicnksr2干扰质粒,并与腺病毒载体(pAdEasy-1质粒)共同转化E.coli BJ5183感受态细菌,产生重组腺病毒载体.用Pac Ⅰ酶切线性化的回收质粒,转染293A细胞,包装腺病毒颗粒,在倒置显微镜下观察细胞病理性变化(Cell pathological effect,CPE),用TCID50法测定病毒颗粒的浓度,并初步观察病毒感染PC12细胞对目的基因的干扰效率.经酶切鉴定、测序证实成功构建小鼠cnksr2基因干扰腺病毒载体.包装的腺病毒浓缩悬液滴度为3.98×107 PFU/mL.cnksr2在大鼠及小鼠中具有保守性,该病毒能在大鼠来源的PC12细胞中成功表达,并且对cnksr2基因干扰效率达50%以上.结论:成功构建了小鼠cnksr2基因的干扰腺病毒载体.
Abstract:
To construct a recombinant adenovirus vector expressing small RNA against mouse cnksr2 gene,the interferce primer were designed according to cnksr2 gene sequence,which was cloned into pshuttle-H1 vector with Bgl Ⅱ and Hind Ⅲ restriction site.E.coli BJ5183 sensitive bacterias were cotransfected with lined vector cutted by Pme I enzyme and adenovirus vector pAdEasy-1.The obtained recombinant adenovirus vector was cutted with Pac Ⅰ enzyme,then adenovirus was obtained in 293A cells transfected with lined recombinant adenovirus plasmids.The titer of virus was measured based on the appearing of CPE and was determined by TCID50 assay.The interference efficiency was caculated.The recombinant adenovirus vector targeting to cnksr2 gene was constructed as evidenced by DNA sequencing and identification of enzyme digestion,which titer of concentrated virus was 3.98×107 PFU/mL.The expression of endogenous cnksr2 mRNA in PC12 cells was specificially shutdown by the recombinant adenovirus vector with an efficiency above 50%.The interference of cnksr2 gene was successfully constructed.

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相似文献/References:

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　Zhao Yinjuan,Lai Shanshan,Li Chaojun,et al.Construction and Identification of GGPPS Gene Interference Adenovirus[J].Journal of Nanjing Normal University(Natural Science Edition),2013,36(03):104.

备注/Memo

备注/Memo:
收稿日期:2012-10-12.
基金项目:国家自然科学基金(31171306)、江苏省自然科学基金(BK2011568).
通讯联系人:薛斌,博士,副教授,研究方向:细胞生物学.E-mail:xuebin@nju.edu.cn
更新日期/Last Update: 2013-09-30