新冠病毒相关蛋白TMPRSS2结构与功能的生信分析及表达载体构建

Bioinformatics analysis on structure and function and expression vector construction of SARS-CoV-2 related protein TMPRSS2

目的人源TMPRSS2是一种跨膜丝氨酸蛋白酶,本文对该蛋白结构与功能进行系统生物信息学分析,优化密码子并构建原核表达载体,以探究其参与SARS-CoV-2侵染宿主细胞的分子机制。方法利用分子克隆技术构建重组表达载体pET-22b-TMPRSS2;采用Protparam、NetPhos3.1、Blast、Clustal X2和MEGA7.0等分析工具对TMPRSS2蛋白的同源性、功能位点、亚细胞定位、三维结构和进化特点等进行生信分析。结果原核表达质粒构建正确;TMPRSS2蛋白属于中等分子量蛋白,由492个氨基酸残基构成,理论等电点为8.12,分子消光系数为118145 L·mol^(-1)·cm^(-1),半衰期30 h;TMPRSS2有15个潜在的糖基化位点,49个可能的磷酸化位点,无信号序列,是一种跨膜的亲水性蛋白。此外,该蛋白有13个潜在的B细胞表位及7个T细胞表位。二级结构分析显示,Random coil在TMPRSS2蛋白中占比最高(0.4533),其次是Extended strand(0.2520)。序列对比和进化分析显示,与人源TMPRSS2序列一致性最高且亲缘关系最近的是Pan troglodytes,其次是Gorilla。结论人源TMPRSS2蛋白在进化上保守,功能重要。本研究结果有助于揭示TMPRSS2蛋白的结构和作用机理,为新冠肺炎的诊疗提供思路,加速了靶向TMPRSS2蛋白的新型药物研发进程。

Aim Human TMPRSS2 is a transmembrane serine protease.In this paper,the structure and function of the protein were systematically analyzed by bioinformatics,the codon was optimized and the prokaryotic expression vector was constructed to explore the molecular mechanism of SARS-CoV-2 infecting host cells.Methods The recombinant expression vector pET-22b-TMPRSS2 was generated by molecular cloning technology.The homology,functional sites,subcellular localization,three-dimensional structure and evolutionary characteristics of TMPRSS2 protein were systematically analyzed by using analytical tools such as Protparam,NetPhos3.1,Blast,Clustal X2 and MEGA7.0.Results The prokaryotic expression plasmid was constructed correctly;TMPRSS2 belongs to medium molecular weight protein,which is composed of 492 amino acid residues.The theoretical isoelectric point is 8.12,the molecular extinction coefficient is 118145 L·mol^(-1)·cm^(-1),and the half-life is 30 h;TMPRSS2 has 15 potential glycosylation sites and 49 possible phosphorylation sites.It is a transmembrane hydrophilic protein without signal sequence.In addition,the protein has 13 potential B-cell epitopes and 7 T-cell epitopes.Secondary structure analysis showed that random coil accounted for the highest proportion of TMPRSS2 protein(0.4533),followed by extended strand(0.2520).Sequence comparison and evolutionary analysis showed that the highest sequence consistency and closest genetic relationship with human TMPRSS2 was Pan troglodytes,followed by gorilla.Conclusions Human-derived TMPRSS2 protein is evolutionarily conserved and functionally important.The results of this study can help to reveal the structure and mechanism of action of TMPRSS2 protein,provide ideas for the diagnosis and treatment of COVID-19,and accelerate the research and development process of new drugs targeting TMPRSS2 protein.