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目的：在大肠杆菌中克隆及高效表达绿色荧光蛋白基因（ＧＦＰ－Ｓ６５Ｔ）。方法：ＰＣＲ法克隆ＧＦＰ－Ｓ６５ＴｃＤＮＡ并改造其两端的限制性内切酶位点，构建重组原核表达载体ｐＲＳＥＴ－ＧＦＰＳ６５Ｔ。结果：在ＩＰＴＧ诱导条件下，ＧＦＰ－Ｓ６５Ｔ的表达量占细菌总蛋白量的１５％以上。细菌培养物及其超声上清在长波紫外线的照射下，发出明亮的绿色荧光。结论：ＧＦＰ－Ｓ６５Ｔ可以作为原核细胞中的报告基因，用标准的长波紫外线即可检测其表达。
Objective: To clone and express green fluorescent protein (GFP-S65T) gene in Escherichia coli. Methods: GFP-S65T cDNA was cloned by PCR and the restriction endonuclease site was modified at both ends to construct recombinant prokaryotic expression vector pRSET-GFPS65T. Results: Under the induction of IPTG, the expression of GFP-S65T accounted for more than 15% of the total bacterial protein. Bacterial cultures and their supernatants fluoresce bright green when exposed to long-wave UV light. Conclusion: GFP-S65T can be used as a reporter gene in prokaryotic cells and its expression can be detected by standard UVA.