Flag-p107真核表达载体的构建及其在ECA109细胞表达及鉴定

Identification and expression of flag tag on the N-terminal of p107 and the flag-p107 expression detection in ECA109 cells

目的构建N端含flag标签的p107的真核表达载体,及其在EC109细胞表达的鉴定。方法设计引物在p107蛋白N端增加flag标签,通过PCR扩增获得3×flag-p107cDNA片段,将此片段插入真核表达质粒CMV-MCS-SV40-Neomycin中;再与质粒CMV-MCS-SV40-Neomycin连接,将所得的融合基因进行PCR、酶切及DNA测序验证正确后,利用电转染将其转染至ECA109细胞中;利用Western-Blotting检测融合蛋白CMV-MCS-3flag-p107-SV40-Neomycin的表达情况。结果重组真核表达载体构建正确,该重组质粒能在ECA109细胞中表达。结论成功构建真核表达载体CMV-MCS-3flag-p107-SV40-Neomycin并建立了其在ECA109细胞的表达。

Objective The eukaryotic expression vector construction that containing the flag tag on the N-terminal of p107,and the flag-p107 expression detection in ECA109 cells.Methods Primers were designed to add the p107 protein N-terminal with flag tag,3 ＆#215; flag-p107 cDNA fragments was obtained by PCR amplification.This fragment was inserted into the eukaryotic expression vector CMV-MCS-SV40-Neomycin;the constructed fusion gene was subjected to PCR,enzyme digestion and DNA sequencing to verify the corrected cloning.Using of electric transfection to transfect the construct into ECA109;utilizing western blotting to detect the fusion protein CMV-MCS-3flagp107-SV40-Neomycinexpression.Results the recombinant eukaryotic expression vector was constructed correctly,and the recombinant plasmid was expressed in ECA109 cells.Conclusion The eukaryotic expression vector CMV-MCS-3flag-p107-SV40-Neomycin is constructed and established its expression in cells ECA109.