CAJ | 학술논문

为开发以绿色荧光蛋白（GFP）为基因的原核表达载体，根据 NCBI 基因序列设计引物，通过 PCR 扩增获得GFP 编码基因，经限制性核酸内切酶消化后将 GFP 定向克隆至原核表达载体 pMAL－c2X 中 malE 基因下游的 EcoRⅠ与 Hind Ⅲ位点之间，与 malE 基因融合为一个表达框。重组产物转化 E．coli TB1感受态细胞，在添加有50 mg/mL AMP、10μmol/L IPTG 的 LB 平板上于37℃培养12～16 h 后放置于25～30℃的培养箱中继续培养4～6 h，365 nm UV 照射下挑取转化克隆，经扩大培养后用 IPTG 诱导 GFP 的表达并用 SDS－PAGE 检测表达效率。结果表明，融合在麦芽糖结合蛋白下游的 GFP 编码基因可以在宿主细胞中有效表达，在365 nm UV 照射下，可以直接从加有50 mg/mL AMP、10μmol/L IPTG 的 LB 平板上挑取发绿色荧光的重组克隆，SDS－PAGE 分析结果显示其扩大培养物经 IPTG 诱导后可高效表达 GFP。该结果为进一步构建以 GFP 为标记基因取代抗生素筛选标记的原核表达载体提供了切实的试验依据。
위개발이록색형광단백（GFP）위기인적원핵표체재체，근거 NCBI 기인서렬설계인물，통과 PCR 확증획득GFP 편마기인，경한제성핵산내절매소화후장 GFP 정향극륭지원핵표체재체 pMAL－c2X 중 malE 기인하유적 EcoRⅠ여 Hind Ⅲ위점지간，여 malE 기인융합위일개표체광。중조산물전화 E．coli TB1감수태세포，재첨가유50 mg/mL AMP、10μmol/L IPTG 적 LB 평판상우37℃배양12～16 h 후방치우25～30℃적배양상중계속배양4～6 h，365 nm UV 조사하도취전화극륭，경확대배양후용 IPTG 유도 GFP 적표체병용 SDS－PAGE 검측표체효솔。결과표명，융합재맥아당결합단백하유적 GFP 편마기인가이재숙주세포중유효표체，재365 nm UV 조사하，가이직접종가유50 mg/mL AMP、10μmol/L IPTG 적 LB 평판상도취발록색형광적중조극륭，SDS－PAGE 분석결과현시기확대배양물경 IPTG 유도후가고효표체 GFP。해결과위진일보구건이 GFP 위표기기인취대항생소사선표기적원핵표체재체제공료절실적시험의거。
The green fluorescent protein(GFP)exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range and has no toxicity on host cells .In cell and molecular biology,the GFP is extensively used as a reporter or detection.In this paper,the GFP encoding sequence was proliferated by PCR under the direction of the primers designed according to the sequence obtained from NCBI website .Then the PCR product was inserted downstream the malE(maltose-binding protein)encoding gene between the EcoRⅠ and Hind Ⅲ sites after restric-tion endonuclease digestion,by which the gfp was fused with malE into one open reading frame.The recombinant prokaryotic expression vector of GFP was employed to transform the competent cells of E.coli TB1.The transforma-tion mixture was cultured at 37 ℃ for 12 -16 h on the LB plates with 50 mg/mL AMP and 10 μmol /L IPTG,and then cultured at 25 -30 ℃ for 4 -6 h on the same plate.The expression of GFP in TB1 was analysed by UV detec-tion prior to SDS-PAGE.The GFP encoding gene infused dowmstream the malE gene can be expressed with green fluorescent excited by 365 nm UV under which the recombinant TB1 clone can be screened conveniently .Further a-nalysis of SDS-PAGE showed that the GFP encoding gene was over expressed in E.coli host TB1.This result showed that the prokaryotic expression vector of GFP was successfully constructed and the malE fusion GFP could be ex-pressed in high-level,which strongly supported the construction of the prokaryotic fusion expression vector with GFP as reporter and detection marker .