【作者】 刘哲民； 李贵阳； 莫照兰； 牟海津；

【Author】 Liu Zhe-min 1 , Li Gui-yang 2 , Mo Zhao-lan 2 , Mou Hai-jin 1* ( 1 College of Food Science and Engineering, Ocean University of China, Qingdao 266003 China; 2 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071 China)

【机构】 中国海洋大学食品科学与工程学院； 中国水产科学研究院黄海水产研究所；

【摘要】 [目的]以具有产kappa-卡拉胶酶能力的菌株Zobellia sp.M2为试验菌株,设计引物获取kappa-卡拉胶酶全基因,构建表达载体HTA-KC及HTA-KC-SIG,在大肠杆菌BL21(DE3)plysS诱导表达可溶性蛋白。分析信号肽与重组酶胞外分泌能力的关系,并进行重组酶分离纯化及其酶学性质的研究。[方法]扩增Zobellia sp.M2的16SrRNA序列,应用MEGA4.0进行进化树分析,并结合NCBI所收录kappa-卡拉胶酶基因序列所属菌株与Zobelliasp.M2的亲缘关系选择引物设计模板,应用Primer-Blast设计引物扩增包含有kappa-卡拉胶酶基因序列片段,对序列进行分析。设计带有限制酶切位点BamH1、XhoI的引物扩增全基因,构建克隆载体pUCm-KC和pUCm-KC-SIG,确认序列无误后构建表达载体HTA-KC及HTA-KC-SIG,分别转入BL21(DE3)plysSIPTG诱导表达,DNS法检测胞外酶活的同时采用Ni-亲和层析法分离纯化带有his-tag标签的重组蛋白,SDS-PAGE进行鉴定。[结果]得到一种新的kappa-卡拉胶酶基因,与现有序列最大相似度为86%。克隆载体确认序列后构建表达载体HTA-KC及HTA-KC-SIG,并成功诱导表达。DNS法测胞外酶活,结合SDS-PAGE结果显示,原卡拉胶酶基因信号肽在重组菌种发挥明显作用,重组蛋白以可溶并具有活性形式表达。该实验为进一步解析重组酶酶学性质,提高重组酶的产量以及实现酶的大量生产等研究奠定了基础。更多还原

【Abstract】 [Objective] A novel kappa-carrageenase gene was cloned from a marine bacterium, Zobellia sp. M2. On the basis of analyzing the full gene sequence, expressing plasmid HTA-KC and HTA-KC-SIG were constructed and the soluble recombined protein was overexpressed in BL21(DE3)plysS. Furthermore, the relationship between the secretion capacity of recombined enzyme and signal peptide sequence was studied, as well as the purification and identification of the recombined kappa-carageenase. [Methods] An optimal template for amplifying the target gene was selected based on the result of Neighbour-joining tree analysis of 16S rRNA with MEGA 4.0. After getting the full gene sequence with a primer pair designed by Primer-Blast, two primer pairs with restriction sites BamH1 and XhoI were designed for the construction of expressing plasmids named HTA-KC and HTA-KC-SIG, respectively, then the recombined protein was induced by IPTG in competent cell BL21(DE3)plysS. DNS method was applied to detect the extracellular enzyme activity, meanwhile, recombined enzyme was purified with Ni sepharose 6FF columns and further identified by SDS polyacrylamide gel electrophoresis (SDS-PAGE). [Results] We successfully discovered a new kind of kappa-carrageenase gene whose maximum similarity was 86% compared with existing kappa-carrageenase gene sequences. Moreover, both expressing vectors HTA-KC and HTA-KC-SIG were structured and transferred into BL21(DE3)plysS, followed by the process of overexpressing soluble recombined proteins. Besides, the recombined kappa-carrageenase secreted outer cell was soluble and activated. We draw the conclusion that the function of signal peptide sequence in BL21(DE3)plysS is obvious by analyzing the extracellular enzyme activity and SDS-PAGE result. In summary, this experiment lays the foundation on further explaining enzymology properties and improving the production of cloning enzyme.更多还原