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目的利用基因重组技术构建原核质粒表达耐甲氧西林金黄色葡萄球菌细胞膜20肽重组蛋白(SA0587),为进一步研究其生物学作用和构建小鼠Ig A肾病动物模型提供重要条件。方法以SA0587的序列为参照,设计出SA0587基因,构建p ET28a-SA0587-GFP重组质粒,以NcoⅠ/XhoⅠ为酶切位点进行双酶切鉴定,同时将符合预期的单克隆进行基因测序,将重组质粒转化至大肠杆菌BL21(DE3)表达菌中;挑取单菌落培养活化后,用诱导剂异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达目的蛋白SA0587;通过SDS-PAGE电泳及Western blot实验鉴定目的蛋白。结果成功构建了重组质粒并进行双酶切鉴定和基因测序,重组质粒大量诱导表达目的蛋白后镍胶纯化,通过SDSPAGE电泳及Western blot实验鉴定目的蛋白为SA0587。结论实验表达和鉴定了SA0587,为后期Ig A肾病小鼠模型的建立提供必要条件。
Objective To construct recombinant prokaryotic plasmids expressing methicillin-resistant Staphylococcus aureus cell membrane 20 peptide recombinant protein (SA0587) by using gene recombination technology, and to provide important conditions for further study of its biological role and construction of mouse IgA nephropathy animal model. Methods Based on the sequence of SA0587, the SA0587 gene was designed and constructed. The recombinant plasmid pET28a-SA0587-GFP was constructed and double-digested with NcoⅠ / XhoⅠ. Meanwhile, the expected single gene was sequenced. Recombinant plasmids were transformed into E. coli BL21 (DE3) -expressing bacteria. After single colony culture, the target protein SA0587 was induced by IPTG induction. SDS-PAGE electrophoresis and Western blot to identify the target protein. Results Recombinant plasmids were successfully constructed and double enzyme digestion and gene sequencing were performed. Recombinant plasmids were induced to express nickel protein gel in large quantities. The target protein was identified as SA0587 by SDSPAGE electrophoresis and Western blot. Conclusion SA0587 was expressed and identified experimentally, which provided the necessary conditions for the establishment of mouse model of IgA nephropathy.