CD36抗原缺失Ⅰ型4种碱基突变及蛋白结构生物信息学分析

Four Base Mutations of CD36 Antigen Deletion TypeⅠand Protein Structure Analyzed by Bioinformatics

目的研究血小板CD36抗原缺失Ⅰ型基因突变对其蛋白结构及功能的影响,了解CD36抗原缺失Ⅰ型基因突变与蛋白结构、功能的关系。方法对血小板CD36抗原缺失Ⅰ型的DNA进行PCR扩增exon3~14等12个外显子的片段并测序。整理获得的DNA序列与CD36基因野生型序列比对,确认12个外显子的起止点并拼接成exon3~14外显子的cDNA序列。用MEGA5.04软件分析cDNA序列的错义突变或同义突变;利用SOPMA进行蛋白二级结构的预测,结合JPred4的辅助,我们能够更准确地揭示蛋白质的构象特征。而借助SWISS-MODEL的空间结构预测能力,我们可以有效推断出氨基酸链的三维构型。Mupro、SDM、CUPSAT、mSCM、DUET、Dynamut预测蛋白突变前后稳定性变化,PROVEAN预测对蛋白功能的影响,PyMOL和LigPlot+修饰蛋白空间结构预测图。结果共检出4种基因突变E4(275).E12(1156).E14(1409)C>T和E6(538)T>C碱基突变,4种突变均导致蛋白结构改变,稳定性降低,3种c.T92M,c.W180R,c.R386W突变对功能有不良影响,5LGD模型和突变后蛋白的配受体间的相互作用基本无变化。结论CD36抗原缺失Ⅰ型碱基突变通过改变蛋白结构进而降低蛋白的稳定性,其中c.T92M,c.W180R,c.R386W突变对蛋白功能产生负面影响。

Objective To study the effect of platelet CD36 antigen deletion type I gene mutation on its protein structure and function,and to understand the relationship between CD36 antigen deletion type I gene mutation and protein structure and function.Methods The DNA of platelet CD36 antigen deletion type I was amplified by PCR and sequenced.The obtained DNA sequence was aligned with the wild-type sequence of the CD36 gene,the start and end points of 12 exons were confirmed,and the cDNA sequence of exon 2 to 14 was spliced.Missense and synonymous mutations in cDNA sequences were analyzed with MEGA5.04 software.Psipred,SOPMA and JPred4 were used to predict the secondary structure of the protein,SWISS-MODEL to predict the spatial structure of the amino acid chain,Mupro,SDM,CUPSAT,mSCM,DUET,Dynamut to predict the stability change of the protein before and after mutation,PROVEAN to predict the effect on protein function,PyMOL and LigPlot+modified protein spatial structure prediction map.Results A total of 4 gene mutations E4(275).E12(1156).E14(1409)C>T and E6(538)T>C base mutations were detected.All 4 mutations lead to changes in protein structure and reduced stability.3 kinds of c.T92M,c.W180R,c.R386W mutations have adverse effects on the function,the 5LGD model and the interaction between the mutant protein's ligand receptor basically unchanged.Conclusion CD36 antigen deletion type I base mutation reduces the protein stability by changing the protein structure,among which c.T92M,c.W180R,c.R386W mutations have a negative impact on protein function.