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ppGalNacT-10cDNA。构建pMD19T-GalNacT-10-ORF和pMD19T-GalNacT-10-antisense,鉴定及测序证实cD-NA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,Westernblot法检测ppGalNacT-10蛋白的表达。结果:pcDNA3.1-GalNacT-10-ORF和pcDNA3.1-GalNacT-10-an-tisense包含大小、序列正确的pp GalNacT-10片段;ppGalNacT-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ppGalNacT-10基因正义和反义真核表达载体。
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.
出处
《郑州大学学报（医学版）》
CAS
北大核心
2012年第6期756-758,共3页
Journal of Zhengzhou University(Medical Sciences)
基金
广东省自然科学基金资助项目S2011040003098
