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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。
Objective To clone the open reading frame of the Ac-fmp-1 gene of the female adult C. elegans and analyze the sequence structure, functional characteristics and physical and chemical properties of the encoded protein and construct an expression vector in vitro. Methods The full-length open reading frame of Ac-fmp-1 gene was cloned by reverse transcription polymerase chain reaction and TA cloning. The coding sequence of Ac-fmp-1 gene and its coding were analyzed by online tools such as NCBI website and ExPaSy bioinformatics tools Amino acid physical and chemical properties, structure and function; using cloning technology targeted cloning prokaryotic expression vector pET-30a-c (+), construct Ac-fmp-1 gene recombinant expression plasmid. Results The open reading frame (ORF) of Ac-fmp-1 gene was amplified and cloned. The sequence of this gene was 1 251bp in length and was 99.6% identical to the coding sequence of Acfmp-1 gene in GenBank. It encoded 417 Amino acids. ProtParam predicted that the gene encoding protein instability in solution, poor conservative, no transmembrane region found. The secondary structure of PHD method predicted that the protein was α-helical and contained a zinc finger structure at the 346-404 amino acid residues. Signal P software predicted that the protein had no signal peptide and had strong hydrophilicity . The amino acid sequence of this gene contains 16 potential B cell epitopes. The constructed prokaryotic expression vector pET-Ac-fmp-1 was successfully inserted into Ac-fmp-1 gene successfully. Conclusion Ac-fmp-1 gene was successfully amplified and cloned. The encoded protein may be an important antigenic component of C. elegans, and may be used as a vaccine candidate molecule and drug target for GNA. Constructed prokaryotic expression vector for in vitro expression, which laid the foundation for the study of Ac-fmp-1 function.