南方农业学报2018，Vol.49Issue(4)：628-634,7.DOI:10.3969/j.issn.2095-1191.2018.04.02
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
Gene prokaryotic expression and specific activity determination of β-glucan synthase KRE9 in Candida tropicalis
摘要
Abstract
[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.关键词
β-葡聚糖合成酶(KRE9)/热带念珠菌/原核表达/纯化/比活力Key words
β-glucan synthase(KRE9)/Candida tropicalis/prokaryotic expression/purification/specific activity分类
农业科技引用本文复制引用
刘政伟,李瑞芳,张瑞玲..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[J].南方农业学报,2018,49(4):628-634,7.基金项目
国家自然科学基金项目(31071922,31572264) （31071922,31572264）
河南省科技攻关项目(162102310404) （162102310404）
