猪纤维蛋白原样蛋白1的克隆表达与生物信息学分析

Cloning,Expression and Bioinformatics Analysis of Swine Fibrinogen-like Protein

为了研究猪纤维蛋白原样蛋白1(FGL1)在免疫抑制性猪病发病机制中的作用,并为相关研究积累材料,提取猪肝脏组织总RNA,采用RT-PCR扩增猪FGL1基因,利用相关生物信息学软件分析猪FGL1的基本理化性质、亲-疏水性、信号肽、磷酸化位点、跨膜结构、二级和三级结构及其潜在的B细胞表位等,并进一步将目的基因插入pcDNA3.1/V5-HisB构建重组载体,转染293T细胞进行真核表达,然后采用Western blot和IFA对表达的目的蛋白进行鉴定。结果显示,猪FGL1基因长951bp,与牛FGL1序列同源性最高(91.4%),与原鸡FGL1序列同源性最低(67.0%);猪FGL1由316个氨基酸组成,理论等电点6.16,为亲水性的非跨膜稳定蛋白;有25个磷酸化位点,氨基端25个氨基酸组成其信号肽;其二级结构主要由α螺旋、延伸链、β转角及无规则卷曲组成;在其第82-309个氨基酸残基之间存在纤维蛋白原相关结构域;Western blot和IFA结果均显示,目的蛋白在转染的293T细胞中获得表达,并具有良好的反应原性。研究结果可以为猪FGL1的相关研究提供参考。

In order to study the role of swine fibrinogen-like protein 1 in the pathogenesis of immunosuppressive disease and accumulate materials for the related research,total RNA was extracted from fresh swine liver tissues and swine FGL1 gene was amplified using RT-PCR.Then the basic physicochemical properties,hydrophilic and hydrophobic properties,signal peptide availability,phosphorylation site,transmembrane structure,secondary and tertiary structures and potential B cell epitopes of swine FGL1 were analyzed using bioinformatics method.Further,recombinant plasmid was constructed by inserting target fragment to the eukaryotic expression vector pcDNA3.1/V5-HisB.293 Tcells were transfected with the recombinant vector and the expression of target gene was detected using Western blot method and IFA.The results showed that swine FGL1 gene was 951 bp long,and this sequence had the highest homology with cow FGL1(91.4%),and the lowest homology with raw chicken FGL1(67.0%).Swine FGL1 is composed of316 amino acid,with the theoretical isoelectric point of 6.16,which is a hydrophilic non-transmembrane stable protein.It has 25 phosphorylation sites,and the 25 amino acids at amino terminal make up its signal peptide.Its secondary structure mainly includes of alpha helix,extension chain,beta angle and irregular curling.The protein has 31 potential B cell epitopes,and it has a fibrinogen related domains at the 82-309 amino acid residues.The results of Western blot and IFA indicated swine FGL1 was expressed in 293 Tcells.The results can provide a reference for the related research of swine FGL1.