广州管圆线虫雌性成虫肌蛋白-1基因的克隆及生物信息学分析

发布时间：2018-04-16 23:02

本文选题：广州管圆线虫 + 雌性成虫肌蛋白-　；参考：《中国病原生物学杂志》2013年10期

【摘要】：目的对广州管圆线虫雌性成虫肌蛋白-1(Ac-fmp-1)基因的开放读码框进行克隆,分析序列结构、功能特征及编码蛋白的理化性质,并构建体外表达载体。方法采用逆转录聚合酶链反应和T-A克隆方法克隆Ac-fmp-1基因的开放读码框全长;运用NCBI网站和ExPaSy等生物信息学在线工具分析Ac-fmp-1基因编码序列及其编码氨基酸的理化性质、结构及功能;采用基因克隆技术定向克隆至原核表达载体pET-30a-c(+),构建Ac-fmp-1基因重组表达质粒。结果扩增和克隆了广州管圆线虫Ac-fmp-1基因开放读码框序列,此序列大小为1 251bp,与GenBank数据库中Acfmp-1基因的编码序列同源性为99.6%,编码417个氨基酸。ProtParam预测该基因编码蛋白在溶液中不稳定、保守性差,未发现跨膜区域。PHD法二级结构预测该蛋白质分子为α-螺旋型,于氨基酸残基346~404位点处含有一个锌指结构,Signal P软件预测该蛋白分子无信号肽序列,具有较强的亲水性。该基因编码蛋白的氨基酸序列中,含有16个潜在的B细胞抗原表位。构建的原核表达载体pET-Ac-fmp-1成功定向插入了Ac-fmp-1基因。结论成功扩增和克隆了广州管圆线虫Ac-fmp-1基因,编码蛋白可能是广州管圆线虫的重要抗原成分,可作为广州管圆线虫病的疫苗候选分子和药物靶标。构建了用于体外表达的原核表达载体,为进行Ac-fmp-1功能研究奠定了基础。
[Abstract]:Objective to clone the open reading frame of myoprotein-1 (Ac-fmp-1) gene of female adult nematode Angiostrongylus andanalyze the sequence structure functional characteristics and physicochemical properties of the encoded protein and construct the expression vector in vitro.Methods the full-length open reading frame of Ac-fmp-1 gene was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) and T-A cloning, and the physicochemical properties of the encoding sequence of Ac-fmp-1 gene and its encoded amino acids were analyzed by using bioinformatics tools such as NCBI website and ExPaSy.The structure and function were cloned into prokaryotic expression vector pET-30a-c (pET-30a-c) by gene cloning technique, and the recombinant expression plasmid of Ac-fmp-1 gene was constructed.Results the open reading frame sequence of Ac-fmp-1 gene of Angiostrongylus cantonensis was amplified and cloned. The size of the sequence was 1251 BP, which was 99.6 homology with the coding sequence of Acfmp-1 gene in GenBank database. ProtParam predicted that the encoding protein of this gene was unstable in solution.The second structure of the protein was predicted to be 伪-helical by transmembrane region. PhD method, and a zinc finger structure signal P software was used to predict the signal peptide sequence of the protein molecule at the site of 346 ~ (404) amino acid residues. The protein had strong hydrophilicity.The amino acid sequence of the gene encoding protein contains 16 potential B cell epitopes.The prokaryotic expression vector pET-Ac-fmp-1 was successfully inserted into Ac-fmp-1 gene.Conclusion the Ac-fmp-1 gene of Angiostrongylus cantonensis was successfully amplified and cloned. The encoded protein may be an important antigen component of Angiostrongylus cantonensis and can be used as a vaccine candidate and drug target for Angiostrongylus cantonensis.The prokaryotic expression vector was constructed for in vitro expression, which laid a foundation for the study of Ac-fmp-1 function.
【作者单位】：南方医科大学附属深圳市妇幼保健院中心实验室;中山大学中山医学院寄生虫学教研室;
【分类号】：R383.19

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