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

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

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1110 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（MB-SP）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。

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 pre-formed 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 pro-tein 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 bio-logical function in E.coli.These data laid a foundation for further studies of EhV99B1-Sp on regulation of virus-host interaction and its application.