B淋巴母细胞呈递来源于乙肝病毒聚合酶和X蛋白的HBV多肽质谱分析  

Mass spectrometry of HBV peptides derived from hepatitis B virus polymerase and X protein presented by B lymphoblastoid cells

作　　者：李佳琪 汝佳秋 鞠晓梅 郭梦蕊 曹馨阳 张淑云[1] LI Jiaqi;RU Jiaqiu;JU Xiaomei;GUO Mengrui;CAO Xinyang;ZHANG Shuyun(Department of Clinical Laboratory,Scientic Research Center,the Second Affiliated Hospital of Harbin Medical University,Harbin 150086,China;The Second Affiliated Hospital of Xiamen Medical College,Xiamen 361021,China;The Third Affiliated Hospital of Changchun University of Traditional Chinese Medicine,Changchun 130022,China)

机构地区：[1]哈尔滨医科大学附属第二医院科研实验中心,哈尔滨150086 [2]厦门医学院附属第二医院,厦门361021 [3]长春中医药大学附属第三临床医院,长春130022

出　　处：《中国免疫学杂志》2025年第2期424-432,I0001-I0029,共38页Chinese Journal of Immunology

基　　金：吴阶平医学基金会临床科研专项资助基金(320.6750.18230)。

摘　　要：目的:应用质谱分析技术及生物信息学技术分析人永生化B淋巴细胞(BLCLs)呈递的来源于Pol和X蛋白的HBV多肽,筛选出有效的T细胞表位,为治疗性疫苗的开发奠定基础。方法:构建含1.2倍HBVC2亚型的全基因组表达质粒,将其通过电转法转染至BLCLs,LC-MS/MS法分离鉴定HBV多肽,生物信息学预测多肽序列的致敏性、抗原性、毒性、HLA分子亲和力及多肽HLA-Ⅰ类复合物与特异性T细胞的结合能力,并检索已报道序列。结果:对5株携带HBVC2亚型表达重组体的BLCLs(BLCLs-1~BLCLs-5)裂解液中的HBV多肽进行分析,成功分离鉴定出141条序列长度不少于8个氨基酸残基(≥8 aa)的多肽,其中133条来自Pol,8条来自X蛋白。通过对141条HBV多肽进行致敏性、抗原性和毒性分析,筛选出50条具有抗原性、无毒性和致敏性的HBV多肽(其中47条来自Pol,3条来自X蛋白),用于与HLA-Ⅰ、Ⅱ类分子的亲和力分析,以及多肽HLA-Ⅰ类复合物与特异性T细胞结合能力的预测。在这些多肽中,37条与HLA-Ⅰ、Ⅱ类分子相应基因型有亲和力。最终获得了37条具有抗原性、无毒性和致敏性,且与HLA-Ⅰ、Ⅱ类分子IC_(50)<500 nmol/L的多肽,这些多肽与相应基因型有亲和力,但未见报道,可作为潜在表位继续探索。最后通过对141条多肽进行株内株间共同、包含、重叠或邻近序列分析,形成了15个HBV多肽热点核心区域,有7条核心区域序列与相应基因型限制的具有抗原性、无毒性和致敏性,IC_(50)<500 nmol/L的多肽亲和力核心序列重叠不少于8个氨基酸,可以优先用于候选T细胞表位进行后续研究。结论:成功分离鉴定出来源于Pol和X蛋白的多肽,筛选出潜在的T细胞表位。Objective:To apply mass spectrometry as well as bioinformatics techniques to analyze HBV peptides derived from Pol and X proteins presented by human immortalized B lymphocytes(BLCLs),and to screen for effective T-cell epitopes,which lays the foundation for the development of therapeutic vaccines.Methods:The group has constructed a genome-wide expression plasmid containing 1.2-fold HBVC2 isoforms and transfected it into BLCLs by electro-transfection,isolated and identified HBV peptides by LC-MS/MS,bioinformatically predicted peptide sequences in terms of sensitization,antigenicity,toxicity,HLA molecular affinity,and the ability of peptide HLA-classⅠcomplex to bind to specific T cells,and retrieved reported sequences.Results:HBV peptides from lysates of five immortalized B cells(BLCLs-1 to BLCLs-5)carrying HBVC2 subtype-expressing recombinants were analyzed,and 141 peptides with sequence lengths of no less than 8 amino acid residues(≥8 aa)were successfully isolated and identified,of which 133 were derived from Pol,and 8 from X proteins.The 141 HBV peptides were analyzed for sensitization,antigenicity and toxicity,and 50 antigenic,non-toxic and sensitizing HBV peptides(47 from Pol and 3 from X protein)were screened for affinity analysis with HLA-classⅠandⅡmolecules and for prediction of the binding ability of the peptide HLA-classⅠcomplexes to specific T cells.Among these peptides,37 had affinity to the corresponding genotypes of HLA-classⅠandⅡmolecules.Finally,we obtained 37 peptides with antigenic,nontoxic,and sensitizing properties with IC_(50)<500 nmol/L to HLA-classⅠandⅡmolecules,which have affinitied to the corresponding genotypes but have not been reported,and can be continued to be explored as potential epitopes.Finally,15 HBV peptide hotspot core regions were formed by intra-and inter-strain common,contained,overlapping or neighboring sequence analysis of 141 peptides,with 7 core region sequences with antigenic,nontoxic,and sensitizing properties restricted to the corresponding genotypes.P

关 键 词：HBV 多肽 Pol蛋白 X蛋白 质谱 生物信息学 

分 类 号：R392.1[医药卫生—免疫学]