一种新的海洋微藻病毒丝氨酸蛋白酶基因的克隆表达及其活性分析

李丽华; 邱健健; 蔡艺钦; 于鹏; 刘静雯

doi:10.3969/j.issn.0253-4193.2014.08.011

一种新的海洋微藻病毒丝氨酸蛋白酶基因的克隆表达及其活性分析

doi: 10.3969/j.issn.0253-4193.2014.08.011

集美大学食品与生物工程学院, 福建厦门 361021;福建省高校食品微生物与酶工程技术研究中心, 福建厦门 361021

基金项目: 国家海洋公益性行业专项（201305027）。

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出版历程
- 收稿日期: 2013-11-14
- 修回日期: 2013-12-19

Cloning,expression and activity analysis of a novel serine protease from marine Coccolithovirus

College of Food and Biological Engineering, Jimei University, Xiamen 361021, China;Research center of food microbiology and enzyme engineering thchndogy of Fujian province, Xiamen 361021, Chine

摘要

摘要: 从海洋球石藻Emiliania huxleyi病毒EhV99B1的基因组中克隆了丝氨酸蛋白酶（Sp）基因（GenBank登录号：KC161207），对该基因的开放阅读框（ORF）进行系统的生物信息学分析，并在大肠杆菌中融合表达，通过亲和层析法获得了纯化的重组Sp。结果表明：EhV99B1-Sp基因的ORF为1 110 bp，编码368个氨基酸，蛋白相对分子质量为39.5 kDa；该基因片段与GenBank中EhV86-Sp的同源性很高，核苷酸及其对应的氨基酸序列同源性分别为95%和97%，而与其他物种Sp序列的同源性仅为28%~32%，说明其可能是丝氨酸蛋白酶家族中的一个新成员；预测的二级结构特征显示EhV99B1-Sp的蛋白结构域中具有典型的LTAGHC（组氨酸活性位点区域）和 AICNGDSGGPLF（丝氨酸活性位点区域）两个丝氨酸蛋白酶催化活性位点的氨基酸基序，是一个两次跨膜蛋白；将该基因在大肠杆菌中进行低温诱导表达，得到分子量为60 kDa的重组蛋白，经鲤鱼肌肉丝氨酸蛋白酶（MBSP）抗体检测证实为Sp，且重组蛋白在大肠杆菌细胞中具有明显的生物学活性。本研究结果为进一步探讨EhV99B1-Sp在病毒与宿主相互作用过程中的调节作用及其功能与应用奠定基础。
- 海洋球石藻病毒 /
- 丝氨酸蛋白酶 /
- 基因克隆 /
- 重组表达 /
- 活性分析
Abstract: A serine protease (Sp) gene (GenBank accession number: KC161207) was cloned from the genome of marine microalgal Emiliania huxleyi virus (Coccolithovirus) EhV99B1 isolate. Bioinformatic analysis was preformed on its open reading frame and the recombinant protein was expressed in E. coli and purified by affinity chromatography. Results showed that the length of the ORF of EhV99B1-Sp was 1 110 bp,which encoded a protein of 386 amino acids with a molecular mass of 39.5 kDa and pI of 6.255. EhV99B1-Sp shared a high sequence similarity with EhV86-Sp,between whom the similarities of nucleotide sequences and deduced amino acid sequences were 95% and 97%,respectively. However,the sequence similarity between EhV99B1-Sp and serine protease from other organisms was only 28% to 32%. Sequences analysis suggested that EhV99B1-Sp might be a new member of the serine protease family. Secondary structure prediction showed that EhV99B1-Sp protein contained two motifs of typical serine protease catalytic active sites LTAGHC (histidine active site) and AICNGDSGGPLF (serine active site),and two transmembrane domains. A 60 kDa recombinant protein of EhV99B1-Sp was expressed in E. coli and confirmed by myofibril-bound serine proteinase (MBSP) antibody. The recombinant protein had normal biological function in E. coli. These data laid a foundation for further studies of EhV99B1-Sp on regulation of virus-host interaction and its application.
- Emiliania huxleyi virus (EhV) /
- serine protease (Sp) /
- gene clone /
- recombinant expression /
- activity analysis

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