应用ELISPOT方法筛选确定尼帕病毒融合糖蛋白和附着糖蛋白的H-2 d限制性T细胞表位

Screening and identification of H-2d-restricted T cell epitopes in fusion and attachment glycoproteins of Nipah virus by ELISPOT

目的:在小鼠中筛选和确定尼帕病毒(Nipah virus,NiV)融合糖蛋白(fusion glycoprotein,F)和附着糖蛋白(attachment glycoprotein,G)特异性的H-2 d限制性T细胞表位。 方法:本研究基于NiV F和G蛋白全序列设计合成多肽库(单肽长为15个氨基酸,前后肽重复10个氨基酸),使用表达NiV F和G蛋白的DNA疫苗肌肉注射免疫BALB/c小鼠,通过矩阵设计将F和G抗原全序列肽库分别混合成肽池,取免疫后小鼠脾细胞进行IFN-γ ELISPOT检测,确定肽库中特异反应的优势表位肽。结果:F肽库筛选到12条特异性优势肽,第56条多肽产生的反应最强;G肽库筛选到4条特异性优势肽,第72条多肽产生的反应最强。结论:本研究使用IFN-γ ELISPOT方法筛选和确定了12条NiV F抗原特异性和4条NiV G抗原特异性H-2 d限制性优势T细胞表位,为NiV免疫学与疫苗研究提供了参考。

Objective To screen and identify H-2d-restricted T cell epitopes in fusion(F)and attachment(G)glycoproteins of Nipah virus(NiV)in mice.Methods The complete peptides(single peptide contains 15 amino acids,and 10 amino acids were repeated in the front and back peptides)derived from F and G antigens were mixed into peptide libraries.BALB/c mice were immunized with DNA vaccines expressing NiV F and G proteins alone and in combination.The full sequence peptide libraries of F and G antigens were mixed into peptide pools by matrix design,and spleen cells of immunized mice were collected and analyzed by IFN-γELISPOT assay to detect the dominant H-2d-restricted epitope peptides.Results Twelve dominant H-2d-restricted peptides were screened from the F protein-specific peptide library and the 56th peptide produced the strongest reaction.Four dominant peptides were screened from the G protein-specific peptide library and the 72nd peptide produced the strongest reaction.Conclusions In this study,12 F antigen-specific and 4 G antigen-specific H-2d restricted dominant T cell epitopes of NiV were screened and identified by IFN-γELISPOT,which could provide reference for immunological analysis of NiV and vaccine research.