C端带His标签重组ScFv真核表达载体的构建及鉴定

Construction and identification of ScFv eukaryotic expression vectors with His-tag at C terminal

目的:构建表达抗p185且含His标签的ScFv的真核表达载体。方法:通过PCR自pcDNAH520C9ScFv-hIL-2质粒中获得H520C9ScFv片段,将此片段插入真核表达质粒pcDNA3.1(+)中;设计引物在ScFv片段C端增加His标签和凝血酶识别序列,再与质粒pcDNA3.1(+)连接,将所得的融合基因进行限制性内切酶EcoR I、HindⅢ双酶切及琼脂糖凝胶电泳鉴定,并测定目的序列。结果:成功将H5200C9ScFv片段、His标签和凝血酶识别序列插入真核表达质粒pcDNA3.1(+)中。结论:成功构建了真核表达载体pcDNA3.1-His-ScFv,为下一步在真核细胞中表达和纯化抗p185单抗奠定基础。

Objective：To construct the ScFv eukaryotic expression vector with His-tag at C-terminal which possessed anti-p185 properties. Methods： The H520CgsFv fragment was derived from the pcDNA-H520CgScFv- hIL-2 plasmid by polymerase chain reaction and inserted into the eukaryotic expression plasmid pcDNA3.1 （ ＋ ）. The primers were designed to allow for addition of the His label and the thrombin-recognizing sequence to the C- terminal of ScFv, which entailed insertion into the eukaryotic expression vector pcDNA3.1 （ ＋ ）. The fusion gene was subject to EcoR I and Hind Ⅲ restriction enzyme digestion and agarose gel electrophoresis for sequencing of the targeted fragment. Results： The H520CgsFv fragment, His label and the thromhin-recognizing sequence have been successfully inserted into the eukaryotic expression vector pcDNA3.1 （ ＋ ）. Conclusion： The successful construction of the eukaryotic expression vector peDNA3.1-His-ScFv provides a solid basis for further research on expression and purification of anti-p185 monoclonal antibodies in eukaryotie cells.