绿色荧光蛋白基因克隆及其表达培养基的筛选

Gene Cloning and Expression Medium Screening of Green Fluorescent Protein(GFP)

为应用PET15b-GFP观察细胞内部结构等功能奠定基础,研究绿色荧光蛋白(Green Fluorescent Protein,GFP)的基因克隆及其在不同种培养基条件下不同种感受态大肠杆菌细胞中的表达状况,通过克隆提纯PET15b-GFP质粒,将已插入单链抗体3E3和152的重组质粒pET15b-3E3-GFP和pET15b-152-GFP,运用转化的方法将重组质粒分别导入感受态的大肠杆菌细胞中(BL21,B cell,K12),在不同培养条件下使用异丙基硫代半乳糖苷(IPTG)诱导GFP基因进行表达,筛选出表达最好的并改良表达能力最弱表达条件。结果表明:重组质粒pET15b-152GFP-BL21在液态LB培养基中30℃、0.4mmol/L IPTG、180r/min诱导条件下培养显色最显著,在固态富集酵母培养基中30℃,1mmol/L诱导条件下培养显色最显著;表达能力最弱的重组质粒pET15b-152-GFP-B cell在富集酵母液体培养基中25℃、180r/min、0.4mmol/L IPTG的条件下诱导培养表达绿色荧光最显著;pET15b-152-GFP-B cell在0 mmol/L、0.1mmol/L、0.4mmol/L和1mmol/L IPTG的诱导条件下表达绿色荧光无显著差异。

To lay the foundation for the application of PET15 b-GFP to observe the internal structure of cells and other functions,the gene cloning of Green Fluorescent Protein(GFP)and its expression in different kinds of competent E.coli cells under different medium conditions were studied,the authors purified the PET15 b-GFP plasmid by cloning,inserted the recombinant plasmids pET15 b-3 E3-GFP and pET15 b-152-GFP into the single-chain antibody 3 E3 and 152,and use the transformation method to introduce the recombinant plasmids into competent E.coli cells(BL21,B cell,K12),and use isopropylthiogalactoside(IPTG)to induce the expression of GFPgene under different media conditions,and select the best expression medium and improve the weakest expression conditions.Results:The recombinant plasmid pET15 b-152 GFP-BL21 gained the most significant color rendering in liquid LB medium at conditions of 30℃,0.4 mmol/L IPTG,and 180 r/min.The recombinant plasmid pET15 b-152 GFP-BL21 expressed in solid enriched yeast medium at 30℃,1 mmol/L induced the most significant coloration;The weakest recombinant expression plasmid pET15 b-152-GFP-B cell performed the most significant in enriched yeast broth at conditions of 25℃,0.4 mmol/L IPTG,180 r/min.pET15 b-152-GFPB cell had no significant difference in expression of green fluorescence under the induction conditions of0 mmol/L,0.1 mmol/L,0.4 mmol/L and 1 mmol/L IPTG.