摘要
Mx蛋白是一类由I型干扰素(IFN)诱导表达的抗病毒蛋白。以PolyI∶C诱导的草鱼(Ctenopharyngodon indellus)肾细胞(CIK cells)为材料,抽提细胞总RNA,通过RT-PCR的方法扩增出Mx基因cDNA全序列,连接至pMD20-T载体上,构建重组质粒pMD20-T-Mx,并测序进行序列分析。用限制性内切酶从克隆的重组质粒pMD20-T-Mx上切下Mx基因,插入到质粒pEGFP-C1中,构建真核表达载体pEGFP-C1-Mx。结果显示,该基因cDNA全序列为1 921 bp,阅读框共编码627个氨基酸,分子量约为71.1 ku,理论等电点pI为8.33,其编码的Mx蛋白具有脊椎动物Mx蛋白共有的结构特征:一个三联体GTP结合区域和一个发动蛋白家族的典型结构特征序列。生物信息学分析表明,草鱼Mx基因所编码的蛋白质以亲水区域为主,具有丰富的B细胞抗原位点,但无明显的核定位序列,空间结构包括N端的GTP酶结构域(GTPase domain)、中间的中央相互作用结构域(central interactive domain,CID)和C端的GTP酶效应结构域(GTPase effector domain,GED)。同源性分析显示,草鱼Mx蛋白氨基酸序列和其他物种的相似性为45.1%～99.7%。此外还成功构建了含草鱼Mx基因cDNA全序列的真核表达载体pEGFP-C1-Mx。研究亮点:首次通过干扰素诱生剂PolyI∶C诱导草鱼肾细胞的方法克隆得到了草鱼抗病毒基因Mx cDNA全序列,并且借助生物信息学软件对其编码的氨基酸进行了序列分析,还成功构建了真核表达载体pEGFP-C1-Mx,为进一步研究草鱼Mx蛋白的生物学活性及其在抗鱼类病毒性疾病中的应用奠定基础。
Mx protein is one of the classes of antiviral proteins induced by type I interferon （IFN）. A fulllength cDNA sequence of Mx gene from grass carp （Ctenopharyngodon indellus） was amplified using reverse transcription polymerase chain reaction （RT-PCR） with total RNA extracted from Ctenopharyngodon indellus kidney （CIK） cells treated With PolyI： C and cloned into pMD20-T vector named pMD20-T-Mx, and sequenced. The recombinant plasmid pMD20-T-Mx and eukaryotic expression vector pEGFP-C1 were cleaved by restriction endonuclease, Mx gene full-length cDNA sequence linkaged with pEGFP-C1. The data indicate that grass carp Mx gene full-length eDNA sequence has 1921 nucleotides, encoding 627 amino acid residues of the mature peptide with a molecular weight of about 71.1 ku and a theoretical isoelectric point of 8.33. The grass carp Mx protein contains a triparitite guanosine-5＇-Triphosphate （GTP）-binding motif and a dynamin family signature sequence, which are conserved in all IFN-indueed Mx proteins of the vertebrates. Bioinformatics analysis reveals that the protein encoded by Mx gene in grass carp is hydrophilic in most regions and has rich B cell antigenicity positions without obvious nuclear localization signal sequence. The dimensional structure of the grass carp Mx protein includes GTPase domain in the amino terminus region, central interactive domain （CID） in the middle and GTPase effector domain （GED） in the earboxyl terminus region. Comparative analysis shows that the Mx protein amino acid sequence of grass carp shares similarity of 45.1% - 99. 7% to those of other species, pEGFP-C1-Mx, which contains Mx gene full-length cDNA sequence and EGFP gene, has been constructed.
出处
《上海海洋大学学报》
CAS
CSCD
北大核心
2012年第4期502-508,共7页
Journal of Shanghai Ocean University
基金
浙江省大学生科技创新活动计划(新苗人才计划)项目(2010R425022)
浙江省自然科学基金(Y3090561
Y3110432)
湖州市自然资金项目(2008YZ01)
关键词
草鱼
MX基因
克隆
序列分析
载体构建
grass carp
Mx gene
cloning
sequence analysis
vector construction
