Sequence analysis of DNA methyltransferase gene from Largemouth bass ulcerative syndrome virus and its expression in prokaryote | 大口黑鲈溃疡综合症病毒甲基转移酶基因序列分析与原核表达

Chino. 为了对新分离到的大口黑鲈溃疡综合症病毒(Largemouth bass ulcerative syndrome virus, LBUSV)的特征作进一步了解，促进LBUSV、DFV(Doctor fish virus)和LMBV(Largemouth bass virus)这类病毒的深入研究，本文克隆了LBUSV DNA 甲基转移酶(DNA MTase)基因约200 bp的保守核心片段，用基因组步移的方法扩增到了核心片段的侧翼序列，获得了DNA MTase基因的编码区全长序列。序列分析表明，DNA MTase基因开放阅读框共663 bp (GenBank登录号：GU256634)，编码220个氨基酸。结构域分析显示，LBUSV DNA MTase蛋白具有4个保守的DNA MTase结构域Ⅰ、Ⅳ、Ⅵ和Ⅷ，以及辅酶结合位点、底物结合位点和DNA结合位点等DNA MTase活性位点，其中的脯氨酸-脯氨酸-半胱氨酸三肽是潜在的催化位点。另外，本文还根据DNA MTase基因开放阅读框设计引物，PCR扩增和酶切后插入到pBV220载体中，经转化大肠杆菌(Escherichia coli)DH5α，温度诱导和SDS-PAGE检测，重组菌在分子量25 kD处有一条特异蛋白带，重组蛋白约占菌体总蛋白的30%。研究结果推测，该基因编码的蛋白具有DNA甲基化的功能，该病毒的分类地位为虹彩病毒科蛙病毒属。

Inglés. In order to further reveal the characteristics of Largemouth bass ulcerative syndrome virus (LBUSV) isolated recently, and further study the group of LBUSV, DFV (Doctor fish virus) and LMBV (Largemouth bass virus), the full-length DNA methyltransferase (DNA MTase) gene was analyzed and expressed using prokaryotic system. A about 200 bp core fragment was amplified and sequenced, and the full-length of DNA MTase was identified by genome walking. The open reading frame (ORF) of DNA MTase gene was 663 bp encoding 220 amino acids (GenBank accession No.GU256634). Motif analysis indicated that LBUSV DNA MTase protein contained blocks Ⅰ, Ⅳ, Ⅵ and Ⅷ, and cofactor binding sites, substrate interacting sites and DNA binding sites were also found in LBUSV DNA MTase, and the proline-proline-cysteine tripeptide was proposed catalytic site. Additionally, the primers were designed according to DNA MTase ORF, and the PCR products were inserted in vector pBV220 and transformed to host Escherichia coli DH5α. After temperature inducement and SDS-PAGE analysis, the recombinant expression bacteria produced a special protein about 25 kD in molecular weight. The proportion of recombinant protein in total bacterial protein was about 30%. Characteristic analysis of DNA MTase gene showed that the predicted protein may play a role of DNA methyltransferase, and confirmed that the taxonomic status of LBUSV is genus Ranavirus of the family Iridoviridae.