HPV16 E5蛋白免疫优势肽段分析及其多克隆抗体的制备

Prediction of immunodominant peptide of HPV16 E5 protein and preparation of polyclonal antibody

目的预测人乳头瘤病毒16型(human papillomavirus 16,HPV16)E5蛋白的免疫优势肽段,并制备其多克隆抗体。方法以人HPV16 E5蛋白全长氨基酸序列为基础,采用Hopp&Woods的亲水性方案、Zimmerman极性参数、Jameson-Wolf抗原指数方案和Emini表面可及性方案及抗原性指数(antigenic index,AI)等生物信息学方法,综合分析E5蛋白免疫优势肽段。通过免疫日本大耳白兔进行E5蛋白免疫优势肽段的免疫原性验证并制备多克隆抗体;进一步采用ELISA法和间接免疫荧光试验分别检测多克隆抗体的效价及特异性。结果HPV16 E5蛋白N-端1~17氨基酸序列(MTNLDTASTTLLACFLL)为免疫优势肽段,可诱导兔产生高水平的E5蛋白特异性IgG抗体,抗体滴度随免疫次数增加而升高;间接细胞免疫荧光试验结果显示,制备的多克隆抗体可特异性识别TC-1-E5细胞胞浆内表达的E5蛋白。结论HPV16 E5蛋白氨基酸肽段(MTNLDTASTTLLACFLL)具有良好的免疫原性;成功制备了HPV16 E5蛋白多克隆抗体,为进一步研究HPV16 E5蛋白的生物学特性奠定了基础。

Objective T o predict the immunodominant peptide of human papillomavirus(HPV)16 E5 protein and prepare its polyclonal antibody.Methods Based on the full-length amino acid sequence of human HPV16 E5 protein,bioinformatics methods such as the Hopp&Woods hydrophilicity scheme,Zimmerman polarity parameter,JamesonWolf antigen index scheme and Emini surface accessibility scheme were combined with the secondary structure of E5 and its flexible region to comprehensively analyze the immunodominant fragment of E5.Japanese white rabbits were immunized with the E5 protein immunodominant peptide to verify the immunogenicity and prepare the polyclonal antibody.The titer and specificity of the prepared polyclonal antibody were determined by ELISA and indirect immuno-fluorescence assay(IFA)respectively.Results The amino acids 1~17(MTNLDTASTTLLACFLL)at N-terminus was the immunodominant peptide of HPV16 E5 protein,which induced high level of E5 protein-specific IgG antibody in rabbits.The antibody titer increased with the increasing doses.IFA showed that the prepared polyclonal antibody specifically recognized the E5 target protein expressed in the cytoplasm of TC-1-E5 cells.Conclusion The amino acid peptide(MTNLDTASTTLLACFLL)of HPV16 E5 protein showed good immunogenicity.The HPV16 E5 protein-specific polyclonal antibody was successfully prepared,which provided a basis for further study on the biological characteristics of HPV16 E5 protein.