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目的:构建pET-32a(+)-PA重组质粒,实现融合蛋白Trx-PA在大肠杆菌(Escherichia coli)BL21(DE3)中的表达,并获得有生物学活性的PA。方法:采用PCR的方法扩增PA的编码序列,并且与pET-32a(+)载体的Trx编码区融合产生pET-32a(+)-PA重组质粒,将重组质粒转化至感受态E.coliBL21(DE3),经IPTG诱导表达pET-32a(+)-PA,通过SDS-PAGE和Western印迹进行分析。利用Ni2+金属亲和层析纯化表达产物,通过梯度透析进行复性。通过蛋白酶降解和细胞毒性实验分析Trx-PA的生物学活性。结果:成功构建pET-32a(+)-PA重组质粒,并纯化了Trx-PA融合蛋白。SDS-PAGE分析表明融合蛋白相对分子质量大小正确,经纯化复性后,蛋白纯度达到76%。Trx-PA在体内和体外均能被弗林蛋白酶(furin酶)降解。Trx-PA的正确降解是致死因子(lethal factor,LF)进入小鼠巨噬细胞Raw264.7内并产生细胞毒性的基础。结论:重组Trx-PA融合蛋白能在E.coliBL21(DE3)中有效表达,并表现出生物学活性。
OBJECTIVE: To construct the recombinant plasmid pET-32a (+) - PA and express the fusion protein Trx-PA in Escherichia coli BL21 (DE3) and obtain the biologically active PA. Methods: The coding sequence of PA was amplified by PCR and fused with the Trx coding region of pET-32a (+) vector to generate recombinant plasmid pET-32a (+) - PA. The recombinant plasmid was transformed into competent E. coli BL21 DE3), pET-32a (+) - PA was induced by IPTG induction and analyzed by SDS-PAGE and Western blotting. The expressed product was purified by Ni2 + metal affinity chromatography and refolded by gradient dialysis. The biological activity of Trx-PA was analyzed by protease degradation and cytotoxicity experiments. Results: The pET-32a (+) - PA recombinant plasmid was successfully constructed and the Trx-PA fusion protein was purified. SDS-PAGE analysis showed that the relative molecular mass of the fusion protein was correct. After purification and refolding, the protein purity reached 76%. Trx-PA is degraded by furin enzyme both in vivo and in vitro. Correct degradation of Trx-PA is the basis for lethal factor (LF) entry into mouse macrophage Raw264.7 and cytotoxicity. CONCLUSION: The recombinant Trx-PA fusion protein can be efficiently expressed in E. coli BL21 (DE3) and shows biological activity.