甲型流感病毒核壳蛋白在BALB/c小鼠中酶联免疫斑点法表位的筛选及其与CTL表位的相关性研究  

Screening of Enzyme-Linked Immunospot Epitopes of Influenza A Virus Nucleocapsid Protein in BALB/c Mice and Studying on Their Relationship with Cytolytic T Lymphocytes Epitopes

作　　者：王文玲[1] 黄保英[1] 王秀平[1] 谭文杰[1] 阮力[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《生物技术通讯》2008年第3期319-323,共5页Letters in Biotechnology

基　　金：国家高技术研究发展计划项目(2006AA02A203)

摘　　要：目的:利用甲型流感病毒A/Johannesburg/33/94(H3N2)核壳蛋白(NP)全长肽库筛选BALB/c(H-2d)小鼠中NP酶联免疫斑点法(ELISPOT)表位,研究其和细胞毒性T淋巴细胞(CTL)表位的一致性关系,为使用ELISPOT评价流感病毒NP疫苗的细胞免疫效果提供实验依据。方法:以甲型流感病毒A/PR/8/34(H1N1)(PR8)感染BALB/c(H-2d)小鼠后,通过检测T细胞分泌γ-干扰素(IFN-γ)的ELISPOT法和体内CTL法检测NP所诱发的细胞免疫反应,综合分析ELISPOT和CTL表位肽之间的关系。结果:Pep36(NP第141～155位氨基酸残基,SNLNDTTYQRTRALV)和Pep37(NP第145～159位氨基酸残基,DTTYQRTRALVRTGM)可以诱发较强的ELISPOT反应,根据Pep36和Pep37共有序列合成的Pep147-155(NP第147～155位氨基酸残基,TYQRTRALV)可以诱发与这2条多肽相同强度的ELISPOT反应,表明Pep147-155为NP诱发ELISPOT反应的最强表位,体内CTL也表明它是最强的CTL表位;Pep95(NP第377～391位氨基酸残基,STLELRSRYWAIRTR)、Pep96(NP第381～395位氨基酸残基,LRSRYWAIRTRSGGN)和其他表位肽诱发的ELISPOT反应较弱,体内CTL反应也较弱。结论:BALB/c(H-2d)小鼠中,甲型流感病毒NP诱发ELISPOT反应和CTL反应的表位肽高度相关;实验结果为使用ELISPOT评价流感病毒NP疫苗的细胞免疫效果提供了实验依据。Objective： To Screen the enzyme-linked immunospot（ELISPOT） epitopes of influenza A virus nucleocapsid protein（NP） in BALB/c（H-2d） mice by using a series of overlapping synthetic peptides covering full length of the amino acid sequence of NP from influenza A virus A/Johannesburg/33/94（H3N2） strain, and to study their relationship with cytolytic T lymphocytes （CTL） epitopes, then to lay experimental foundation for the evaluation of cellular immune response elicited by influenza A virus NP protein-based vaccine by ELISPOT. Methods： BALB/c（H-2d） mice were infected with influenza A virus A/PR/8/34（H1N1）（PR8） strain, then cellular immune response elicited by NP were detected by mouse IFN-y ELISPOT and in vivo CTL, after that the consistency of ELISPOT epitopes and CTL epitopes of NP were analyzed. Results： Pep36（residues 141-155, SNLNDTrYQRTRALV） and Pep37（residues 145-159, DTrYQRTRALVRTGM） could elicit strong ELISPOT response; Pep147-1s5 （residues 147-155, TYQRTRALV）, synthesized according to the consensus se- quence of Pep36 and Pep37, was able to elicit as strong ELISPOT response as both of them, it suggested that Pep147-155 was the strongest ELISPOT epitope of NP, in vivo CTL assay also suggested that Pep147-155 was the strongest CTL epitope; Pep95（residues 377-391, STLELRSRYWAIRTR）, Pep96（residues 381-395, LRSRYWAIRTRSGGN） and other peptides could elicit both poor ELISPOT response and poor CTL responses. Conclusion： The data indicated that ELISPOT epitopes of influenza A virus NP were highly related with those of CTL in BALB/c（H-2d） mice, and all the experimental results showed that ELISPOT could be used to evaluate cellular immune response elicited by influenza A virus NP-based vaccine.

关 键 词：甲型流感病毒 核壳蛋白 酶联免疫斑点法 体内细胞毒性T淋巴细胞 

分 类 号：R392.1[医药卫生—免疫学]