SARS-CoV-2 M蛋白生物信息学分析及真核表达载体的构建与鉴定

Bioinformatics analysis and construction、identification the Eukaryotic expression vector of SARS-CoV-2 M protein

目的利用生物信息学方法预测新型冠状病毒膜(SARS-CoV-2 M)蛋白的结构与性质,构建SARS-CoV-2 M蛋白真核表达载体,为抗新型冠状病毒药物和疫苗的研发提供一定参考。方法利用NCBI查找SARS-CoV-2 M蛋白的核苷酸序列和氨基酸序列,利用ORF Finder预测SARS-CoV-2 M蛋白的开放阅读框数目;Protparam预测SARS-CoV-2 M蛋白的理化性质;SignalP和TMHMM2.0预测SARS-CoV-2 M蛋白的信号肽与跨膜区;NetPhos、SOPMA和SWISS MODEL预测SARS-CoV-2 M蛋白的磷酸化位点、糖基化位点、二级结构与三级结构;IEDB、Immunomedicine Group、Cell-Ploc 2.0和Uniprot预测分析SARS-CoV-2 M蛋白的T细胞和B细胞表位、抗原决定簇、亚细胞定位及与其相互作用的蛋白。通过合成目的基因后插入到真核表达载体pcDNA3.1(+)中,双酶切鉴定后进行序列测序,将重组质粒转染至Vero细胞中,再以Western blot方法检测该基因编码蛋白的表达情况。结果编码SARS-CoV-2 M蛋白基因全长669 bp,由222个氨基酸组成;其分子式为C1165H1823N303O301S8,相对分子质量为25146.62,理论等电点为9.51,不稳定系数为39.14,脂肪族氨基酸系数为120.86,平均亲水系数为0.446;SARS-CoV-2 M蛋白有5个开放阅读框,无信号肽,3个跨膜区,15个磷酸化位点,1个糖基化位点,二级结构以无规则卷曲为主,占比45.5%;6个B细胞表位,8个Th细胞表位,平均抗原倾向为1.0532,主要与11个蛋白发生相互作用。成功构建了pcDNA3.1(+)-M重组质粒,双酶切鉴定插入片段大小正确,DNA测序结果与SARS-CoV-2 M基因序列同源性为100%,Western blot成功检测出该蛋白在Vero细胞内的表达。结论成功预测了SARS-CoV-2 M蛋白多项生物信息并成功构建pcDNA3.1(+)-M重组质粒,为抗新型冠状病毒药物和疫苗的研发提供了一定参考。

Objective To predict the structure and properties of novel coronavirus membrane protein(SARS-CoV-2 M protein)by bioinformatics method,and to construct the prokaryotic expression vector of SARS-CoV-2 M protein,so as to provide a new target for the development of anti-novel coronavirus drugs and vaccines.Methods NCBI was used to search the nucleotide sequence and amino acid sequence of SARS-CoV-2 M protein.Protparam,ORF Finder,SignalP,TMHMM2.0,NetPhos,NetNGlyc,SOPMA,SWISS MODEL,IEDB,Immunomedicine Group,and Cell-Ploc 2.0 and STRING were used to predicted and analyzed the physicochemical properties,open reading frame,signal peptide,transmembrane region,phosphorylation site,glycosylation site,secondary structure,tertiary structure,T cell and B cell epitopes,antigenic determinant,subcellular localization and interacting proteins of SARS-CoV-2 M protein.The target gene was synthesized and inserted into the eukaryotic expression vector pcDNA3.1(+),identified by double enzyme digestion and sequenced,The recombinant plasmid was transformed into Vero cell,Western blot was used to detect the expression of the protein that encoded by SARS-CoV-2 M gene.Results The total length of SARS-CoV-2 M gene was 669 bp.The SARS-CoV-2 M protein was composed of 222 amino acids.The molecular formula was C1165H1823N303O301S8,molecular weight was 25146.62,and the theoretical isoelectric point was 9.51.The instability coefficient was 39.14,the aliphatic amino acid coefficient was 120.86,and the average hydrophilic coefficient was 0.446.There were 5 open reading frames,no signal peptide,3 transmembrane regions,15 phos-phorylation sites and 1 glycosylation site.The secondary structure was dominated by random coil,accounting for 45.5%,6 B cell epitopes and 8 Th cell epitopes,with an average antigenic propensity of 1.0532,and mainly interacting with 11 proteins.The pcDNA3.1(+)-M recombinant plasmid was successfully constructed,and the size of the inserted fragment was correct by double enzyme digestion.The DNA sequencing results showed 100%homology with the M gene of SARS-CoV-2,the expression of the protein in eukaryotic cells was suc-cessfully detected by Western blot.Conclusion The biological information of SARS-CoV-2 M protein was predicted successfully and the recombinant plasmid pcDNA3.1(+)-M was constructed successfully,which provides a new target for the development of novel coronavirus drugs and vaccines.