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目的检测铜绿假单胞菌噬菌体PaP3末端酶大亚单位与噬菌体基因组末端cos位点的结合能力。方法通过PCR扩增出末端酶大亚单位编码基因tls,经pMD-T18载体克隆至表达载体pQE31上,IPTG诱导表达,获得包涵体蛋白,用包涵体裂解液溶解包涵体,通过Ni-NTA亲和层析,分离出重组目的蛋白rTLS,透析复性后,与生物素标记的基因组末端cos片段进行结合反应,通过EMSA检测DNA滞后现象。结果成功构建了表达载体pQE-tls,获得了纯化的具有生物学活性的重组末端酶大亚单位rTLS,EMSA结果证实结合rTLS后的cos片段与无蛋白加入的对照组相比明显滞后。结论重组噬菌体PaP3末端酶大亚单位在体外可与基因组末端cos片段发生特异性结合,为进一步研究该蛋白的功能奠定了基础。
Objective To detect the binding ability of the Pseudomonas aeruginosa bacteriophage PaP3-terminal large subunit to the cos-site of the phage genome. Methods The tls gene was amplified by PCR and cloned into expression vector pQE31 using pMD-T18 vector. IPTG was used to induce the expression of inclusion body protein. Inclusion body lysate was used to dissolve the inclusion bodies. And chromatographic separation, the target protein rTLS was isolated, dialyzed and refolded, and then bound to biotin-labeled cosmid. The DNA lag was detected by EMSA. Results The expression vector pQE-tls was successfully constructed and the purified rTLS with large biological activity was obtained. The result of EMSA showed that the costimulatory fragment after rTLS binding was significantly delayed compared with the control without protein addition. Conclusion The recombinant phage PaP3-terminal large subunit can specifically bind to cos band at the end of the genome in vitro, which lays the foundation for further study on the function of this protein.