推定铜绿假单胞菌噬菌体PaP3末端酶小亚单位的.docVIP

PAGE PAGE 4 推定铜绿假单胞菌噬菌体PaP3末端酶小亚单位的DNA结合能力检测* 申晓冬1 ，张克斌2，李明1，胡晓梅1 ，周莹冰1 ，陈志瑾1，胡福泉1 （第三军医大学：1基础部微生物学教研室，2新桥医院医学实验技术中心） 摘 要：目的 检测理论推定的铜绿假单胞菌噬菌体PaP3末端酶小亚单位（pap3p01基因）编码蛋白对特异性DNA的结合能力。方法 通过PCR从噬菌体PaP3基因组扩增出pap3p01基因，克隆至表达载体pQE31，转化入大肠杆菌JM109后，IPTG诱导表达目的蛋白，超声裂菌后发现目的蛋白质H6-PaP3P01存在于上清中，进而利用Ni-NTA亲合层析纯化蛋白。利用PCR与酶切方法获取可能含有末端酶小亚单位结合位点的DNA片段，并对其3’端进行生物素标记。最后采用凝胶迁移率改变实验检测H6-PaP3P01的DNA 结合能力。结果 成功构建了pQE-PaP3P01表达载体，获得的融合蛋白H6-PaP3P01表达量较高且全部存在于菌体超声后的上清中。经亲和层析初步纯化及脱盐处理后，H6-PaP3P01可与263bp特异性DNA片段结合。 结论 成功构建并表达了推定的PaP3末端酶小亚单位重组蛋白H6-PaP3P01，并且检测到了该蛋白质对特异性DNA 的结合能力，初步证实了理论推定的正确性，为完善PaP3 关键词：末端酶小亚单位，噬菌体PaP3，EMSA，蛋白表达 Determination of the binding ability to specific DNA sequence of the putative small subunit of the terminase of Pseudomonas aeruginosa bacteriophage PaP3 Abstract: Objective To investigate the binding ability to specific DNA sequence of the putative small subunit of the terminase of Pseudomonas aeruginosa bacteriophage PaP3. Methods The gene of PaP3p01 was amplified from the genome of bacteriophage PaP3 and cloned into expression plasmid pQE31, the recombinant vector pQE31-PaP3p01 was transformed to E. coli JM109, after induction with IPTG, the expressed bacteria was dissolve with lysis buffer, then the tagged protein was purified by Ni-NTA affinity chromatography. The DNA fragment carrying the putative small subunit binding site was gained by PCR and restrictive enzyme cut method, and marked with biotin in 3’-end, finally the DNA-binding ability of the fusion protein H6-PaP3P01 was determined by EMSA. Results The expression plasmid pQE31-PaP3p01 was successfully constructed, the fusion protein H6-PaP3P01 was highly expressed and in the suspended layer. After purified by Ni-NTA affinity chromatography and desalted, the H6-PaP3P01 protein can binding to the specific DNA fragment. Conclusion The fusion protein H6-PaP3P01 was successfully expressed and purified, and it has the specific DNA-binding activity Key words: terminase small subunit; bacteriophage PaP3; EMSA; pro