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目的:构建大肠杆菌-毕赤酵母表达载体Ppic9k-IL3-Linker-PE38KDEL。方法:用PCR的方法扩增所需要的目的片段IL3及PE38KDEL,再通过酶切和连接的方法定向克隆到载体Ppic9k-Linker中,得到融合基因Ppic9k-IL3-Linker-PE38KDEL。重组载体经酶切,菌落PCR鉴定,DNA序列分析插入片段完全正确。结果:经限制性内切酶酶切鉴定,菌落PCR及DNA序列分析表明重组表达载体Ppic9k-IL3-Lin-ker-PE38KDEL构建成功。结论:成功地构建融合基因IL3-PE38KDEL的毕赤酵母表达载体,为后续的蛋白质的表达、纯化及功能研究奠定基础。
Objective: To construct E. coli-Pichia pastoris expression vector Ppic9k-IL3-Linker-PE38KDEL. Methods: The desired fragment IL3 and PE38KDEL were amplified by PCR and cloned into vector ppic9k-Linker by restriction enzyme digestion and ligation to obtain the fusion gene Ppic9k-IL3-Linker-PE38KDEL. Recombinant vector by digestion, colony PCR identification, DNA sequence analysis insert is completely correct. Results: The recombinant plasmid ppic9k-IL3-Lin-ker-PE38KDEL was successfully constructed by restriction endonuclease digestion, colony PCR and DNA sequence analysis. Conclusion: The successful construction of Pichia pastoris expression vector with IL3-PE38KDEL fusion gene lays the foundation for the subsequent study of protein expression, purification and function.