Dhori病毒核蛋白抗原表位的筛选及鉴定

Screening and identification of antigenic epitopes of nucleoprotein in Dhori virus

目的研究鉴定DHOV核蛋白(NP)的最小线性B细胞表位(BCEs),为DHOV的疫苗研制和预防奠定基础。方法本研究针对DHOV-GRT169毒株NP蛋白进行生物信息学分析,采用改良生物合成肽法鉴定其BCEs。首先将DHOV NP蛋白截短为4个片段并鉴定其抗原性,将阳性片段继续截短成相互重叠8个氨基酸的16肽,将16肽序列分别克隆至原核表达载体pXXGST-3中表达,兔抗融合蛋白NP蛋白多克隆抗体作为一抗,用Western blotting检测16肽的抗原性,将检测出的阳性16肽都截短成相互重叠7个氨基酸的8肽,用同样的方法诱导表达及检测。最后用生物信息学方法分析每个BCEs在NP蛋白三维结构中的位置。结果从NP蛋白中最终鉴定出9个BCEs,分别为Enp1(^(3)NPTPKR^(8))、Enp2(^(7)KRAEPGD^(13))、Enp3(^(83)YUFFFL^(88))、Enp4(^(111)NQTLTDE^(117))、Enp5(^(224)QSQKAMLK^(231))、Enp6(^(227)KAMLKQIF^(234))、Enp7(^(418)LNAEFEEY^(425))、Enp8(^(421)EFEEYSKL^(428))、Enp9(^(432)GTGAFYER^(439)),均位于NP蛋白表面。结论这些鉴定的表位将提高对DHOV表位分布和致病机制的认识,为DHOV多表位检测抗原的开发提供基础,也可为DHOV感染与免疫机制研究提供理论依据。

Dhori virus(DHOV)is a new tick-borne virus in China.It can infect large livestock such as sheep,cattle,and camels,and has the potential to harm humans.Therefore,this study identified the minimum linear B-cell epitope(BCE)of the DHOV nuclear protein(NP)to lay a foundation for the development of a DHOV vaccine.In this study,the NP protein of the DHOV-GRT169 strain was analyzed by bioinformatics,and its BCEs were identified by an improved biosynthetic peptide method.First,the DHOV NP protein was truncated into four fragments and its antigenicity was identified.The positive fragments were further truncated into 16 peptides overlapping eight amino acids.The 16 peptide sequences were cloned into a prokaryotic expression vector pXXGST-3 for expression.A rabbit anti-fusion protein NP polyclonal antibody was used as the primary antibody.The antigenicity of 16 peptides was detected by western blotting,and the 16 peptides were truncated into eight peptides overlapping seven amino acids.The expression was induced and detected by the same method.Finally,the position of each BCE in the three-dimensional structure of NP was analyzed by bioinformatics.Nine BCEs were finally identified from NP.These were Enp1(^(3)NPTPKR^(8)),Enp2(^(7)KRAEPGD^(13)),Enp3(^(83)YUFFFL^(88)),Enp4(^(111)NQTLTDE^(117)),Enp5(^(224)QSQKAMLK^(231)),Enp6(^(227)KAMLKQIF^(234)),Enp7(^(418)LNAEFEEY^(425)),Enp8(^(421)EFEEYSKL^(428)),Enp9(^(432)GTGAFYER^(439)),all located on the surface of NP.These identified epitopes will improve the understanding of DHOV epitope distribution and pathogenesis,provide a basis for the development of DHOV multi epitope detection antigen,and provide a theoretical basis for the study of DHOV infection and immune mechanism.