登革病毒非结构蛋白1 氨基端抗原性分析及其检测登革病毒感染动物血清

目的    精确分析登革病毒(dengue virus，DENV)非结构蛋白1（non-structural protein 1， NS1)氨基(N)端抗原性和免疫原性，探讨NS1 N端多肽用于分型检测DENV感染可行性。方法    合成4型DENV 标准株NS1蛋白 N端1-15氨基酸残基序列多肽（D-1 P1、D-2 P1、D-3 P1和D-4 P1），采用ELISA方法检测多肽同DENV NS1 血清型特异性单抗和各型DENV分别免疫小鼠血清的反应性，竞争抑制法进一步验证单抗和对应多肽反应特异性。分析4型DENV 标准株NS1 N端1-15氨基酸残基序列多肽在Pubmed中已报道的各型DENV分离株中的保守性。结果     6株DENV 1型特异性单抗（5A48A3, 5A65A2, 5B29A1, 5B71A8, 5C29A3和5E68A17）特异性的同D-1 P1；6株DENV-2 NS1型特异性单抗（5D21A1、5E19A5、 5A62A12、5A70A4、5E30A5和5E48A15）特异性的同D 2 P1反应。D-1 P1仅识别DENV 1免疫小鼠血清， 其他多肽同各型DENV 1免疫小鼠血清反应无差异。DENV-1、2、3、4 NS1 N端保守性依次为98.96%、89.09%、83.58%和57.81%。结论     DENV-1 NS1 N端是DENV-1血清型特异性优势线性表位，该多肽可用于研制诊断DENV-1感染试剂盒；DENV-2 NS1 N端是DENV-2血清型特异性表位，以上研究有助于DENV亚单位疫苗和DENV感染分型诊断试剂盒的研制。

Precisely mapping the antigenicity and immunogenicity on N-terminal regions of the dengue virus non-structural protein 1 and detecting DENV-immunized animal serum samples using an epitope-based peptide antigen

Objective     To precisely determine the antigenicity and immunogenicity on N-terminal regions of DENV NS1 and develope serodiagnosis of DENV infection using an epitope-based peptide antigen. Methods    The synthesis peptide sequences of N-terminal regions of DENV NS1 were derived from the four serotype DENV standard strains and named D-1 P1, D-2 P1, D-3 P1 and D-4 P1. ELISA was performed to analyze antibody responses to these peptides from monoclonal antibodies (MAbs) with dengue serotype specificity as well as antisera from mice immunized with the dengue virus serotypes. The specific reactivity of peptides with corresponding MAbs was further conformed by competitive inhibition experiments. The degree of conservation and variability of N-terminal regions of DENV NS1 within DENV isolates which were reported in Pubmed were analysised by Bioinformatics. Results    Six DENV-1 serotype-specific MAbs (5A48A3, 5A65A2, 5B29A1, 5B71A8, 5C29A3, and 5E68A17) showed a strong reaction with D-1 P1. Six DENV-2 serotype-specific MAbs (5D21A1, 5E19A5, 5A62A12, 5A70A4, 5E30A5 and 5E48A15) showed a strong reactionwith D-2 P1. D-1 P1 only reacted with all 3 sera from DENV-1-immunized mice. The other peptides were not different from sera from mice immunized with all four dengue virus serotypes. Alignment revealed that the conservation of 100% homology for N-terminal regions of DENV-1,2,3,4 NS1 was 98.96%, 89.09%, 83.58% and 57.81%, respectively.  Conclusions    N-terminal regions of Dengue virus-1 non-structural protein 1 contained an immunodominant serotype epitope and N-terminal regions of Dengue virus-2 non-structural protein 1 contained a serotype epitope. D-1 P1-based peptide antigen will be useful for serologic diagnosis of DENV-1 infection. These novel serotype B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and serodiagnosis assays.