丙型肝炎病毒NS5ab区的B细胞模拟表位的确认  

Identification of HCV NS5ab's B cell mimotopes with phage display random peptide

作　　者：侯利华[1] 杜桂鑫[1] 王海涛[1] 

机构地区：[1]军事医学科学院微生物流行病研究所分子生物学研究室,北京100071

出　　处：《免疫学杂志》2003年第4期265-268,273,共5页Immunological Journal

基　　金：国家自然科学基金重点资助项目 (396 30 0 2 0 )

摘　　要：目的　用噬菌体展示随机肽库研究丙型肝炎病毒 (HCV)NS5ab区的B细胞表位。方法　在原核系统中表达了HCVNS5ab重组蛋白 ,亲和层析纯化血清中抗HCVNS5ab的特异多抗 ,用此多抗来筛选噬菌体递呈随机 12肽库 ,获得HCVNS5ab区的B细胞表位。结果　成功地表达了HCVNS5ab重组蛋白 ,用此蛋白检验了 130份HCV阳性血清 ,阳性率为 36 %。在筛选的噬菌体展示随机肽库后 ,挑取 13个阳性克隆测序 ,有 9个序列不同 ,最终通过噬菌体表位表达的方法确认了 3个表位。Objective To identify the B cell mimotopes of HCV NS5ab with phage display random peptide library. Methods HCV NS5ab gene from the sera of a chronic HCV patient was expressed in E.coli (M15) and purified by Ni-NTA column. The HCV NS5ab protein was coupled with sepharose-4B agarose and the specific polyclonal antibodies against HCV NS5ab protein were obtained from HCV (+) sera by affinity chromatography. The phage display random peptide library was screened by the specific polyclonal antibodies. The peptides including different phage PⅢ protein domain Ⅰ-Ⅱ from the phages was expressed in E.coli (M15) and secreted into the periplasm. The serological assays showed good results with three peptides by ELISA. Results The antigenicity of the recombinant protein was tested by ELISA with 130 HCV (+) sera and 10 HCV (-) sera. The positive percent was 36%. 13 positive clones were obtained by biopanning phage display random peptide library and resulted in 9 different peptide sequences. Three linear epitopes were identified in the NS5ab by expressing the peptides of the phages. Conclusions It is feasible that the phage display random peptide library was screened by the specific polyclonal antibodies for the B cell mimotopes of HCV NS5ab.

关 键 词：丙型肝炎病毒 NS5ab区 B细胞模拟表位 噬菌体展示随机肽库 

分 类 号：R346[医药卫生—基础医学]