目的 在原核细胞和真核细胞中分别构建,表达StayGold绿色荧光蛋白同时融合功能性表达抗生素抗性基因的载体。方法 将StayGold编码序列插入抗生素抗性基因的不同位置,分别构建相应载体。原核载体转化于大肠杆菌DH10B中,真核载体转染于HEK293T细胞中。利用抗生素筛选,探究抗生素抗性基因表达情况。利用荧光显微镜对样本进行观测,检测StayGold表达情况,并与EGFP的荧光强度进行对比。将细菌菌液样本于4℃冰箱放置一个月后重新检测荧光强度,对比融合蛋白StayGold与EGFP的低温稳定性。结果 在所有原核载体中,StayGold均能正常表达。仅有将StayGold插入AmpR的C端（添加连接肽与否）,被转化了对应载体的大肠杆菌DH10B均对氨苄青霉素表现为抗性,同时其荧光强度与对照组EGFP相近,但是,一个月后实验组StayGold的荧光强度高于EGFP。在所有真核载体中,StayGold均能够正常表达。将StayGold插入PuroR或HygroR序列C端,并添加连接肽32AA linker,被转染了对应载体的HEK293T均显示对对应抗生素的耐受。结论 将StayGold插入抗生素抗性基因C端,并添加连接肽,可分别在原核细胞和真核细胞中实现荧光和抗性的功能融合。在大肠杆菌中,StayGold有比EGFP更强的低温稳定性。
Abstract
Objective To construct vectors that express StayGold green fluorescent protein in both prokaryotic and eukaryotic cells, while simultaneously expressing functional fused antibiotic resistance genes. Method The StayGold coding sequence was inserted at different positions within the antibiotic resistance gene to construct corresponding vectors. The prokaryotic vectors were transformed into E. coli DH10B, while the eukaryotic vectors were transfected into HEK293T cells. Antibiotic selection was used to investigate the expression of the antibiotic resistance gene. Fluorescence microscopy was harnessed to observe the samples and detect the expression of StayGold, with its fluorescence intensity compared to that of EGFP. After storing the bacterial culture samples in a refrigerator at 4℃ for one month, the fluorescence intensity was re-measured to compare the low-temperature stability of the fusion proteins StayGold and EGFP. Results In all prokaryotic vectors, StayGold was expressed normally. Only when StayGold was inserted into the C-terminus of the AmpR gene with or without a peptide linker (32 residues, 32AA), the transformed E. coli DH10B exhibited resistance to ampicillin. Moreover, its fluorescence intensity was comparable to that of the control group EGFP. However, after one month, the fluorescence intensity of the experimental group StayGold was higher than that of EGFP. In all eukaryotic vectors, StayGold was also expressed normally. When StayGold was inserted into the C-terminus of the PuroR or HygroR sequence, and a 32AA linker was added, the transfected HEK293T cells showed tolerance to the corresponding antibiotics. Conclusion Inserting StayGold into the C-terminus of the antibiotic resistance gene encoding protein and adding a linker can achieve functional fusion of fluorescence and resistance in both prokaryotic and eukaryotic cells. In E. coli, StayGold exhibits stronger low-temperature stability compared to EGFP.
关键词
StayGold /
原核表达载体 /
真核表达载体 /
融合蛋白
Key words
StayGold /
prokaryotic expression vector /
eukaryotic expression vector /
fusion protein
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基金
国家自然科学基金面上项目“利用FAM72A促进CBE介导的精准基因编辑来修复ABCA4突变”（82271910）
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