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目的构建SLO原核表达质粒,重组蛋白的诱导表达并纯化。方法提取链霉菌溶血素O模板DNA,PCR法扩增slo基因。构建融合表达重组质粒p GEX-6p-1-slo和pet32a-tev-slo,将正确的重组质粒转化至大肠杆菌BL21和大肠杆菌BL21-DE3,使用异丙基硫代βD半乳糖苷(IPTG)诱导表达重组融合蛋白。采用亲和层析纯化重组蛋白,切除标签后,再通过亲和层析纯化,获取SLO重组蛋白。结果 PCR扩增出slo基因,基因片段(1700 bp)与理论一致。经SDSPAGE,蛋白质印迹显示重组蛋白相对分子质量为55 k Da,与数据库中的相对分子质量结果相符。结论成功构建了slo原核表达质粒,表达并纯化了SLO重组蛋白。
Objective To construct the prokaryotic expression plasmid of SLO and induce the recombinant protein expression and purification. Methods Streptomyces hemolysin O template DNA was extracted and amplified by PCR. The recombinant plasmids pGEX-6p-1-slo and pet32a-tev-slo were constructed and transformed into Escherichia coli BL21 and Escherichia coli BL21-DE3 respectively. The recombinant plasmids were transformed with IPTG, Induced expression of recombinant fusion protein. The recombinant protein was purified by affinity chromatography. After the tag was excised, the recombinant protein was purified by affinity chromatography. Results The slo gene was amplified by PCR. The gene fragment (1700 bp) was consistent with the theory. Western blotting showed that the relative molecular weight of recombinant protein was 55 kDa by SDSPAGE, which was in good agreement with the database of relative molecular mass. Conclusion The prokaryotic expression plasmid of slo was successfully constructed and the SLO recombinant protein was expressed and purified.