大肠杆菌分泌表达纳豆激酶及体外溶栓特性分析  

Secret expression of nattokinase in E.coli and its thrombolytic property in vitro

作　　者：叶延欣 李蕾蕾 宋书涵 张杰 刘亚琼 洪军 徐振上 YE Yan-xin;LI Lei-lei;SONG Shu-han;ZHANG Jie;LIU Ya-qiong;HONG Jun;XU Zhen-shang(School of Life Science&Engineering,Henan University of Urban Construction,Pingdingshan 467036,China;Henan Health Food Engineering&Technology Research Center,Henan University of Urban Construction,Pingdingshan 467036,China;School of Bioengineering,Qilu University of Technology,Jinan 250104,China)

机构地区：[1]河南城建学院生命科学与工程学院,河南平顶山467036 [2]河南省健康食品工程技术研究中心,河南平顶山467036 [3]齐鲁工业大学生物工程学院,山东济南250104

出　　处：《河南城建学院学报》2022年第6期79-84,92,共7页Journal of Henan University of Urban Construction

基　　金：国家自然科学基金(31901665)。

摘　　要：以Bacillus subtilis NK4基因组为模板,克隆出纳豆激酶(Nattokinase,NK)前导肽与成熟肽编码基因(NK),并采用同源重组方法将其与已报道的功能肽编码基因Cel或Fe及线性化质粒pET-22b构建出重组表达载体pET22b-Cel-NK与pET22b-Fe-NK,并转化于E.coli BL21(DE3)中进行诱导表达。经IPTG诱导表达后,实现了重组NK在大肠杆菌中的重组表达并具有生物活性,但SDS-PAGE仅检测到重组酶Cel-NK的可分泌表达及其具有酶催化活性,酶活力为(3.36±0.56) IU/mL,体外溶栓实验也表明该重组酶NK具有良好的体外溶栓效果,表明Cel有助于介导蛋白NK可活性分泌表达。Using the Bacillus subtilis NK4 genome as a template,the nattokinase leader peptide and mature peptide-encoding gene(NK) was cloned,and then the recombinant expression vectors pET22b-Cel-NK and pET22b-Fe-NK were constructed by homologous recombination with the reported functional peptide cod gene Cel or Fe and the linearization plasmid pET-22b,and transformed into E.coli BL21(DE3) for induced expression,respectively.After the expression was induced by IPTG,the recombinant nattokinase was expressed in E.coli and it had enzymatic activity.However,SDS-PAGE only detected the secretory expression of the recombinant enzyme Cel-NK and its enzymatic activity was(3.36±0.56) IU/mL.Thrombolytic experiments also showed that the recombinant nattokinase had good activity,indicating that Cel helped to promote the active secretory expression of nattokinase.

关 键 词：纳豆激酶 同源重组 分泌表达 酶催化活性 体外溶栓 

分 类 号：Q939.9[生物学—微生物学]