用噬菌体展示随机12肽库筛选HCV B细胞抗原表位

Selection of antigenic mimics of hepatitis C virus by phage display peptide library

目的用患者血清中抗丙型肝炎病毒(HCV)抗体从噬菌体展示随机12肽库筛选HCV抗原表位.方法用硫酸铵粗提HCV患者血清中Ig后用DEAE-Sephadex A50层析纯化并作为筛选的配基;对噬菌体展示随机12肽库采用配基亲和富集、阴性血清吸收的方法进行生物淘洗;ELISA鉴定筛选克隆的结合特性;测定阳性克隆所携带DNA序列并进行计算机辅助分析.结果三轮生物淘洗后特异性克隆得到了富集.用第3轮淘洗出的26个克隆进行结合实验,其中有3个克隆与HCV患者血清结合力较高而与正常人血清结合能力较弱;序列分析发现2个序列与HCV蛋白第1 082～1 093和1 954～1 965位(1082YHGAGTRTIASP 1093和1954TQLLRRLHQWIS 1965)氨基酸有较高的同源性;计算机辅助分析提示这两个位点具有形成表位的性质.结论获得了两个HCV抗原表位,在HCV感染的检测中具有潜在的应用价值.

Objective To obtain the antigenic epitopes of hepatitis C virus (HCV) by the polyclonal antibodies against HCV using a 12-mer phage peptide library. Methods Polyclonal antibodies of HCV were purified from hepatitis C (HC) patient sera using DEAE-Sephadex A-50 column chromatography. A 12-mer phage peptide library was biopanned for 3 rounds by the purified Abs and normal sera. The purified Abs were selective molecules, and the normal sera were used to absorb the nonspecific phage clones. The HCV specific phage clones were identified using ELISA by comparison of the reactions between the phage clones chosen after the third round biopanning and the patient or normal sera. The result was confirmed by computer assistant analysis. Results After 3 rounds of effective biopanning and ELISA, 3 positive clones were obtained out of 26 clones screened. The amino acid sequences of the 2 positive clones were homogeneous to that of HCV amino acids 1 082-1 093 and 1 954-1 965. Computer assistant analysis also supported the result. Conclusion The peptides displayed by the positive phage can mimic the epitopes of HCV antigen, which provide the potentiality for detecting the HCV infection.