新型人冠状病毒NL63膜蛋白(M)基因的序列分析及其抗原表位预测分析

Sequence analysis and prediction of related B cell epitopes of membrane protein(M)of human coronavirus NL63

目的了解新发现的、与北京地区婴幼儿急性呼吸道感染相关的人冠状病毒HCoV- NL63的主要结构蛋白——膜蛋白(M)的基因特征。方法分别从2份已确认为HCoV-NL63阳性的、取自急性呼吸道感染患儿的标本(BJ3140和BJ8081)中用RT-PCR方法扩增得到其M蛋白的全基因,在对其进行了序列分析的基础上对推导出的M蛋白的抗原表位进行了预测分析。结果BJ3140和BJ8081的M蛋白编码基因全长681bp,编码一个含226个氨基酸的蛋白。BJ3140和BJ8081之间M基因的核苷酸和氨基酸同源性分别为99.1%和99.6%;与HCoV-NL63原型株有很高的核苷酸(99.1%～99.7%)和氨基酸同源性(99.6%和98.7%),而与HCoV-229E的氨基酸同源性为61.3%,与HCoV- OC43和SARS-CoV氨基酸同源性则低于31.0%。BJ3140和BJ8081的M蛋白N端21个氨基酸位于病毒包膜外区,随后是3次跨膜区,最后是较长的包膜内区。不同方法的预测结果显示BJ3140的M蛋白的B细胞表位优势肽段多位于C-末端包膜内区域。结论从北京地区婴幼儿HCoV-NL63感染阳性的呼吸道标本中扩增得到的M蛋白编码基因与HCoV-NL63原型株有很高的同源性,具有与其他冠状病毒M蛋白相似的结构特征。

Objective To characterize membrane protein（M） gene of a novel human coronavirus-NL63 （HCoV-NL63） identified from samples collected from children with acute respiratory tract infections（ARIs） in Beijing. Methods Full length of membrane protein encoding genes, M genes of HCoV-NL63 were amplified from two specimens, BJ3140 and BJ8081, collected from children with acute respiratory infections by reverse-transcriptase and polymerase chain reaction（RT-PCR）. The two PCR amplicons were sequenced after cloning into T vector and the sequences were compared with M genes from two prototype strains and other human coronaviruses in GenBank. B cell epitopes on membrane protein were predicted by bio-informatic software. Results The M genes amplified from samples BJ3140 and BJ8081 were 681 bp in length, encoding protein of 226 amino acids. The identities of nucleotide and amino acid between BJ3140 and BJ8081 were 99.1% and 99.6%, respectively. Compared with the prototypes of HCoV-NIJ53, sequence identifies of BJ3140 and BJ8081 were 99.1%-99.7% at the nucleotide level, 99.6% and 98.7% at the amino acid level, whereas amino acids identifies were 61.3% as compared with HCoV-229E and below 31.0% as compared with HCoV-OCA3 and SARS-CoV. The structure of M proteins of BJ3140 and BJ8081 has three domains： a 21 amino acid N-terminal ectodomain, a triple-spanning transmembrane domain and a long interior C-terminal domain. Majority of the immunodominant B cell epitopes of M protein located in the interior C-terminal region. Conclusion M genes encoding membrane protein amplified from HCoV-NL63 positive samples from children with ARIs had high identities with two prototype swains; M proteins of HCoV-NL63 from Beijing had a structure similar to the M proteins of other coronaviruses.