摘要
【目的】原核表达热带念珠菌(Candida tropicalis MYA-3404)β-葡聚糖合成酶(KRE9),并进行酶比活力测定,为研究其结构和生物学功能提供参考。【方法】提取热带念珠菌DNA,PCR扩增KRE9基因,构建其原核表达载体pET28a-KRE9后转化大肠杆菌(Escherichia coli)BL21(DE3)感受态细胞,通过IPTG诱导表达及组氨酸(His)标签纯化树脂亲和柱纯化获得融合蛋白KRE9,并利用3,5-二硝基水杨酸(DNS)显色法测定其比活力。【结果】克隆获得的KRE9基因全长816 bp,编码271个氨基酸,与参考基因序列(GenBank登录号XM_002547984)的相似性高达99.75%。将其原核表达载体pET28a-KRE9转化大肠杆菌BL21(DE3)感受态细胞,通过诱导表达及纯化获得重组蛋白KRE9,经SDS-PAGE检测,发现其纯度较高,大小约35 k D。Western blotting鉴定结果显示,重组蛋白KRE9能与抗His抗体发生特异性结合。重组蛋白KRE9比活力为9.169×10~4U/mg。【结论】重组蛋白KRE9具有良好的特异性和较高β-葡聚糖合成酶活性,可用于其结构和生物学功能研究。
【Objective】β-glucan synthase KRE9 of Candida tropicalis MYA-3404 was expressed and purified,and its specific activity was analyzed,in order to provide reference for the study on the structure and biological function of β-glucan synthase KRE9.【Method】The DNA of C. tropicalis was extracted,gene KRE9 was amplified by PCR,and prokaryotic expression vector p ET28 a-KRE9 was constructed. Competent cell of Escherichia coli BL21(DE3)was transfered.Fusion protein KRE9 was obtained by induction of IPTG as well as histidine(His)tag purificationof resin affinity chromatography,and its specific activity was determined by 3,5-dinitrosalicylic acid(DNS)colorimetricmethod.【Result】The cloned gene KRE9 was 816 bp in total length,encoding 271 amino acids. The sequencing results showed that gene KRE9 was highly similar to reference genes(GenBank accession number XM_002547984),which reached 99.75%. The prokaryotic expression vector p ET28 a-KRE9 was transformed into competent cell of E. coli BL21(DE3). Then the recombinant protein KRE9 was obtained by induction and purification. SDS-PAGE detection results showed that the recombinant proteins was 35 kDa with high purity.Western blotting identification indicated that there was specific binding between purified recombinant protein KRE9 and His antibody. The specific activity of KRE9 was 9.169×10~4 U/mg.【Conclusion】Recombinant protein vegetable proteinis of fine specificity and high β-glucan synthase activity,and it can be used for its structural and biological function research.
引文
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