热带念珠菌β-葡聚糖合成酶KRE9基因原核表达及其比活力测定

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 kD.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9.169×104U/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-glu-can synthase KRE9.[Method]The DNA of C.tropicalis was extracted,gene KRE9 was amplified by PCR,and prokary-otic expression vector pET28a-KRE9 was constructed.Competent cell of Escherichia coli BL21(DE3)was transfered.Fu-sion protein KRE9 was obtained by induction of IPTG as well as histidine(His)tag purificationof resin affinity chromatog-raphy,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 pET28a-KRE9 was transformed into competent cell of E.coli BL21(DE3).Then the recombinant pro-tein 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 re-combinant protein KRE9 and His antibody.The specific activity of KRE9 was 9.169×104U/mg.[Conclusion]Recombi-nant protein vegetable proteinis of fine specificity and high β-glucan synthase activity,and it can be used for its structural and biological function research.