[1]高媛),刘振华),冯菁华),等.pp GalNacT10基因真核表达载体的构建及表达*[J].郑州大学学报(医学版),2012,(06):756.
 GAO Yuan,LIU Zhenhua,FENG Jinghua,et al.Construction and expression of eukaryotic expression vector for pp GalNacT10 gene[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2012,(06):756.
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pp GalNacT10基因真核表达载体的构建及表达*
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2012年06期
页码:
756
栏目:
论著
出版日期:
2012-11-20

文章信息/Info

Title:
Construction and expression of eukaryotic expression vector for pp GalNacT10 gene
作者:
高媛1)刘振华2)冯菁华1)李晓云1)孙其喆1)张宝1)郑文岭13)马文丽1)#
1)南方医科大学基因工程研究所 广州 510515
2)苏州卫生职业技术学院 苏州 215009
3)华南基因组研究中心 广州 510800
Author(s):
GAO Yuan1)LIU Zhenhua2)FENG Jinghua1)LI Xiaoyun1)SUN Qizhe1)ZHANG Bao1)ZHENG Wenling13)MA Wenli1)
1)Institute of Genetic Engineering,Southern Medical University,Guangzhou 510515
2)Suzhou Health College,Suzhou 215009
3)South China Genome Research Center,Guangzhou 510800
关键词:
pp GalNacT10真核表达载体293T细胞
Keywords:
pp GalNacT10eukaryotic expression vector293T cell
分类号:
R34
摘要:
目的:构建pp GalNacT10基因真核表达载体。方法:采用PCR方法合成含有酶切位点(XbaⅠ、EcoRⅠ)的pp GalNacT10 cDNA。构建pMD19TGalNacT10ORF和pMD19TGalNacT10antisense,鉴定及测序证实cDNA片段大小和序列。双酶切目的载体pcDNA3.1后,与GalNacT10ORF和GalNacT10antisense片段进行连接,构建正义和反义真核表达载体pcDNA3.1GalNacT10ORF和pcDNA3.1GalNacT10antisense,将其分别转染293T细胞,Western blot法检测pp GalNacT10蛋白的表达。结果:pcDNA3.1GalNacT10ORF和pcDNA3.1GalNacT10antisense包含大小、序列正确的pp GalNacT10片段;pp GalNacT10蛋白在转染pcDNA3.1GalNacT10ORF的293T细胞中高表达,在转染pcDNA3.1GalNacT10antisense的293T细胞中表达降低。结论:成功构建了pp GalNacT10基因正义和反义真核表达载体。
Abstract:
Aim:To construct eukaryotic expression vector of pp GalNacT10 gene.Methods:PCR synthesis full length of pp GalNacT10 cDNA containing restriction sites(XbaⅠ,EcoRⅠ).pMD19TGalNacT10ORF and pMD19TGalNacT10antisense were build,and the sequence and size of pp GalNacT10 cDNA fragment were confimed to be correct.After objective vector pcDNA3.1 was digested,it was separately connected with GalNacT10ORF and GalNacT10antisense.Sense and antisense eukaryotic expression vectors pcDNA3.1GalNacT10ORF and pcDNA3.1GalNacT10antisense were separately transfected into 293T cells.Western blot was used to identify the expressed product.Results:The results of digestion confirmed the right length of inserted DNA,which was the same as the pp GalNacT10 cDNA,and pp GalNacT10 protein was highly expressed in 293T cells which were transfected with pcDNA3.1GalNacT10ORF,and low expressed in 293T cells which were transfected with pcDNA3.1GalNacT10antisense.Conclusion:pcDNA3.1GalNacT10ORF and pcDNA3.1GalNacT10antisense have been successfully constructed.

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备注/Memo

备注/Memo:
*广东省自然科学基金资助项目S2011040003098
#通讯作者,女,1964年4月生,博士,教授,研究方向:基因诊断与基因治疗,Email:wenli668@gmail.com
更新日期/Last Update: 2013-07-03