鸡粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因的克隆及生物信息学分析

为了解GM-CSF的基因及蛋白质特性,本研究以pUC-GM-CSF为模板,根据GenBank中登录的GM-CSF序列设计引物扩增GM-CSF基因,随后将其克隆至 pMD18-T载体,并对鉴定正确的重组质粒 pT-GM-CSF进行测序分析,然后对GM-CSF基因进行同源性分析、ORF分析、氨基酸序列分析、疏水性分析、磷酸化位点预测、亚细胞定位分析及二级结构预测。结果表明：该基因ORF的全部长度为453 bp,能够编码150个氨基酸,并且在GM-CSF基因所编码的蛋白质中有4个丝氨酸和3个苏氨酸可能成为蛋白激酶磷酸化的位点。同时,GM-CSF蛋白的最大疏水性为2.267,且有8.91%的可能位于细胞外,有0.8%的可能位于细胞壁,有0.24%的可能位于细胞质。同时,该蛋白质的二级结构主要由无规卷曲、α螺旋和延伸链构成。本研究通过对GM-CSF基因进行生物信息学分析,为探究其免疫增强机制奠定基础。

Cloning and Bioinformatics Analysis of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) Gene

We aim to investigate the gene and protein properties of GM-CSF. In this study, pUC-GM-CSF was used as a template to amplify GM-CSF gene according to the GM-CSF sequence registered in GenBank, and then this gene was cloned into pMD18-T vector. Sequencing was performed for the recombinant plasmid pT-GM-CSF. Bioinformatics tools were used to analyze homology and ORF of GM-CSF gene. Moreover, the amino acid sequence, hydrophobicity/hydrophilicity, phosphorylation site, subcellular localization and secondary structure were analyzed for the encoded protein. The results showed that the total open reading frame of this gene was 453 bp, encoding 150 amino acids. In the protein encoded by GM-CSF, 4 serine residues and 3 threonine residues may be phosphorylated. The maximum hydrophobicity of GM-CSF protein was 2.267. This protein had 8.91% possibility to be located outside the cell, 0.8% possibility to be located on the cell wall, and 0.24% possibility to be located in cytoplasm. The secondary structure of the protein was mainly composed of random coil, α helix and extension chain. These results can lay a foundation for exploring the immune enhancement mechanism of GM-CSF.