摘要
目的将人类Tudor—SN（tudor staphylococcal nuclease）蛋白SN（1～4）基因片段分别定向连入pEGFP-C2质粒，使Tudor—SN蛋白SN各功能片段与绿色荧光蛋白在HeLa细胞内融合表达。方法以重组质粒pSG5-Tudor-SN-flag为模板，PCR法扩增出目的基因，利用EcoRⅠ和SalⅠ双酶切法将目的片段连接到pEGFP—C2载体上，再将构建成功的pEGFP-C2-Tudor—SN-SN（1～4）重组质粒转染入HeLa细胞内，以荧光显微镜及Western印迹法检测绿色荧光蛋白与目的蛋白的融合表达情况。结果以单／双酶切及基因测序法鉴定构建的重组质粒均无误，荧光显微镜及Western印迹结果均检测到绿色融合蛋白的表达。结论重组pEGFP-C2-hTudor—SN-SN（1—4）质粒成功构建并表达。
Objective To examine the construction and expression of the recombinant eukary- otic plasmid pEGFP-C2-hTudor-SN-SN （ 1 - 4 ） . Methods The target gene Tudor-SN （ tudor staphylococcal nuclease） was amplified by PCR, with the recombinant plasmid pSGS-Tudor-SN-flag serving as templates. The target segment was connected to the pEGFP-C2 vector by using the double- enzyme digestion （EcoR Ⅰ and Sal Ⅰ ） . The successfully constructed recombinant plasmids pEGFP- C2-Tudor-SN-SN （ 1 -4） were then transfected into HeLa cells. Fluorescence microscopy and West-ern blotting were employed to detect the expression of green fluorescent prntein-fused target pro- teins. Results The single/dnuble-enzyme digestion and gene sequencing revealed lhat the sequence of the recombinant plasmid was correct. Fluorescence microscopy and Western blotting showed the expression of green fusion prntc, in. Conclusion The recombinant plasmid pEGFP-C2-hTudor-SN-SN （ 1 -4） was successfully constructed and expressed.
出处
《医学分子生物学杂志》
CAS
CSCD
2012年第4期241-245,共5页
Journal of Medical Molecular Biology
基金
国家杰出青年基金项目（No.31125012）,国家高技术研究发展计划资助项目（863计划）（No.2007AA022115）,国家自然科学基金（No.30970582,31100967,31170830）,国家自然科学重大研究计划培育项目（No.90919032）,国家教育部高等学校博士学科点专项科研基金（No.20091202110001）,中国博士后科学基金（No.2011M500529）,天津市教委重点项目（No.2008ZD01）
