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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263 bp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DNA的结合能力,初步证实了理论推定的正确性,为完善PaP3噬菌体包装机制的研究奠定了基础。
Objective To detect the binding ability of the putative Pseudomonas aeruginosa bacteriophage PaP3-terminal small subunit (pap3p01) encoded protein to specific DNA. Methods The pap3p01 gene was amplified by PCR from the phage PaP3 genome and cloned into the expression vector pQE31. After transformed into E. coli JM109, the target protein was induced by IPTG and the target protein H6-PaP3P01 was found in the supernatant. Ni-NTA affinity chromatography purification of protein. The DNA fragments that may contain the small subunit binding site of the terminal enzyme were obtained by PCR and restriction enzyme digestion, and their 3 ’ends were labeled with biotin. Finally, the DNA binding ability of H6-PaP3P01 was tested by gel shift assay. Results The pQE-PaP3P01 expression vector was successfully constructed. The expression level of the fusion protein H6-PaP3P01 was high and all existed in the supernatant after the cells were ultrasonically treated. H6-PaP3P01 can be combined with 263 bp specific DNA fragments after preliminary purification and desalting by affinity chromatography. Conclusion The putative PaP3-terminal small subunit recombinant protein H6-PaP3P01 was successfully constructed and expressed, and its binding ability to specific DNA was detected. The correctness of the theoretical inference was initially confirmed, which lay the foundation for the improvement of the packaging mechanism of PaP3 phage The foundation.