细菌-酵母穿梭表达质粒pGBKT7-MIF的构建及转化

Construction and Transformation of Bacteria-Yeast Shuttle Plasmid pGBKT7-MIF

【目的】构建并转化能在酵母菌AH10 9中表达巨噬细胞游走抑制因子 (MIF)的穿梭表达质粒pGBKT7 MIF。【方法】用RT PCR方法从人T淋巴细胞中扩增MIF基因全外显子片段 ,并定向克隆入细菌 酵母穿梭表达质粒 pGBKT7中 ;用PEG/LiAC法将pGBKT7 MIF转化感受态酵母菌AH10 9。提取转化酵母菌的质粒DNA ,转化大肠杆菌并行质粒的酶切鉴定。【结果】扩增出人MIFcDNA片段 ,限制性内切酶酶切和DNA测序证实获得正确的重组质粒 pGBKT7 MIF ;获得可在色氨酸缺陷培养基 (SD/ trp)上生长含 pGBKT7 MIF的酵母菌克隆。酶切鉴定表明pGBKT7 MIF已转化入酵母菌AH10 9。【结论】成功构建穿梭质粒 pGBKT7 MIF ,并转化入酵母菌AH10 9。

To construct and transform bacteria yeast shuttle plasmid pGBKT7 MIF. The fragment including all exons of macrophage migration inhibitory factor gene was amplified by RT PCR and was inserted into the multiple cloning sites of the shuttle vector pGBKT7. By using PEG/LiAC method, the plasmid pGBKT7 MIF was transformed into yeast AH109.The plasmid isolated from the positive yeast AH109 clone was transformed into E.coli DH5α, which was to be amplified and digested by restrictive endonucleases. The human MIF cDNA was amplified. The restrictive endonuclease digestion and DNA sequencing proved that the plasmid pGBKT7 MIF was obtained. The yeast AH109 clone transformed with pGBKT7 MIF was screened out by the SD/ trp nutritional media. The restrictive endonuclease digestion showed that the plasmid pGBKT7 MIF was transformed into yeast AH109.[Conclusion] The bacteria yeast shuttle plasmid pGBKT7 MIF was successfully constructed and transformed into yeast AH109.