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目的克隆金黄葡萄球菌表面蛋白凝集因子A(ClfA)活性基因,并进行原核表达。方法以金黄葡萄球菌基因组DNA为模板,采用PCR方法扩增ClfA(221-550)活性基因,克隆入载体pET28a(+)中,构建重组表达质粒pET28a-ClfA(221-550),转化感受态大肠杆菌BL21(DE3),IPTG诱导表达,并对表达产物进行SDS-PAGE和Western blot分析。结果PCR扩增出987bp的目的基因片段,重组表达质粒经双酶切证明构建正确。表达产物经SDS-PAGE分析,在相对分子质量约36000处可见目的条带,目的蛋白表达量约占菌体总蛋白的36.76%,且具有良好的反应原性。结论已成功克隆了金黄葡萄球菌ClfA活性基因,并在大肠杆菌BL21(DE3)中表达了目的蛋白。
Objective To clone the ClfA gene of Staphylococcus aureus and express it in prokaryotic cells. Methods The genomic DNA of Staphylococcus aureus was used as template to amplify the ClfA (221-550) active gene by PCR and cloned into vector pET28a (+). The recombinant plasmid pET28a-ClfA (221-550) was constructed and transformed into competent colon Bacillus BL21 (DE3), IPTG induced expression, and the expression product was analyzed by SDS-PAGE and Western blot. Results The 987bp gene fragment was amplified by PCR. The recombinant plasmid was confirmed by double enzyme digestion. The expressed product was analyzed by SDS-PAGE, and the target band was observed at about 36,000 relative molecular mass. The expressed protein of the target protein accounted for about 36.76% of the total bacterial protein, and had good reactionogenicity. Conclusion ClfA gene of Staphylococcus aureus has been successfully cloned and expressed in E. coli BL21 (DE3).