凝胶阻滞试验分析铜绿假单胞菌LexA蛋白与其靶位点的特异性结合

Analysis on the specific combination of the binding target site and LexA protein in pseudomonas aeruginosa by the gel retardation experiments

目的:分析铜绿假单胞菌(pseudom onas aeruginosa,PA)的LexA与其靶位点的特异性结合的特性。方法:以PCR法扩增PA的LexA基因,将其克隆于原核表达载体pET-22b(+),转化大肠埃希菌BL21(DE3),IPTG诱导表达,采用镍离子亲合柱层析和Superdex-75凝胶过滤层析分离和纯化目的蛋白,以含推定LexA结合位点的片段CTGTN27ACAG为探针,用凝胶阻滞试验检测LexA蛋白与探针序列结合的能力及特异性。结果:构建了pET22b-LexA重组质粒,IPTG诱导目的蛋白表达率约为25%,获得纯度大于95%的目的蛋白;CTGTN27ACAG能与LexA蛋白发生特异性结合。结论:推定LexA结合位点CTGTN27ACAG能与PA的LexA蛋白特异性结合,可能是LexA蛋白的结合靶位点。

Aim：To analyze the specific combination of repressor protein LexA and its binding target site of Pseudomonas aeruginosa. Methods： The LexA of PAO1 was ligated with pMD18-T vector, digested with restriction endonuclease NdeI and XhoI, and then cloned into pET22b（ ＋ ）. pET22b-LexA was verified by NdeI,XhoI digestion and sequencing. The recombinant plamid pET22b-LexA was transformed into E. coli BL21 （DE3）. 0. 2mmol/L IPTG was added to induce expression of LexA. The expressed protein was purified by two steps of Ni2 ＋ chelate affinity chromatography and Superdex-75 gel filtration chromatography subsequently. The binding target site of LexA that contains a conservative palindrome sequence of CTGT and ACAG was used as the probe. The interaction of the probe and the LexA was analyzed by the gel retardation experiments. Results： The expected DNA fragment was observed with 1.0% agarose electrophoresis, and the gene of LexA was successfully cloned into pET22b（ ＋ ）, which was confirmed by restriction endonuclease digestion and sequencing. The results of 15% SDS-PAGE showed that the molecular mass of the expressed protein was 22KD, which was consistent with the anticipated one, and the expressed rate was 25%. The expressed protein was found in insoluble form, and the purity of the recombinant protein was up to 95% by two steps of Ni2 ＋ chelate affinity chromatography and gel filtration chromatography subsequently. The gel retardation and competitive inhibition experiments and showed that the LexA can bind with probe specifically. Conclusion： CTGTN27 ACAG, binding site of LexA, may bind specifically with repressor protein LexA.