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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 an eukaryotic expression vector expressing His-tagged ScFv against p185. Methods: H520C9ScFv fragment was obtained from pcDNAH520C9ScFv-hIL-2 plasmid by PCR. The fragment was inserted into eukaryotic expression plasmid pcDNA3.1 (+). The primers were designed to add His tag and thrombin recognition sequence to the C-terminus of ScFv fragment. The plasmid pcDNA3.1 (+) was ligated. The resulting fusion gene was identified by restriction endonucleases EcoR I, Hind III digestion and agarose gel electrophoresis, and the target sequence was determined. Results: H5200C9ScFv fragment, His tag and thrombin recognition sequence were successfully inserted into the eukaryotic expression plasmid pcDNA3.1 (+). Conclusion: The eukaryotic expression vector pcDNA3.1-His-ScFv was successfully constructed, which laid the foundation for the next step to express and purify anti-p185 mAb in eukaryotic cells.