能结合HCV E2蛋白的人CD81模拟肽的筛选与活性鉴定

目的用CD81单克隆抗体筛选噬菌体随机展示十二肽库,以期得到可模拟人CD81功能位点、能抑制HCVE2与其受体—人CD81分子结合的小肽。方法用CD81单抗从噬菌体随机展示十二肽库中筛选人CD81模拟肽,用ELISA鉴定阳性噬菌体克隆;以阳性噬菌体免疫BALB/c小鼠,分析小鼠免疫血清对阳性噬菌体与HCVE2结合的阻断作用;以HCVE2蛋白包被酶标板,检测阳性噬菌体对HCVE2与人CD81分子结合的抑制作用。结果对十二肽库进行3轮筛选后,经ELISA和DNA测序,鉴定出3个(C4,C13和C16)阳性克隆,与人CD81无同源序列。阳性噬菌体克隆C16免疫小鼠血清能阻断该克隆与HCVE2的结合。C16克隆能以剂量依赖的方式竞争性抑制HCVE2蛋白与人CD81的结合。结论用人CD81单抗从噬菌体随机展示肽库中筛选出的阳性噬菌体克隆所编码的小肽在功能上能模拟人CD81与HCVE2的结合活性,能竞争性抑制HCVE2与人CD81分子的结合,在抗HCV药物及疫苗研究中具有潜在应用价值。

Biopanning and Identification of Peptides Capable to Inhibit the Interaction between hCD81 and HCV E2 Protein

Objective To screen for active short peptide sequences from a 12-mer phage display peptide library, that can mimic human CD81 (hCD81) functional domain and inhibit the binding of hepatitis C virus (HCV) envelope glycoprotein E2 with its cellular receptor hCD81 molecule. Method Monoclonal antibody to hCD81 was used to screen the phage displayed library. The screened positive phage clones were then identified, and used to immunize BALB/C mice. The serum from immunized mice was collected and measured for its blocking activity against positive phages and HCV E2 interaction. The inhibitory effect of the positive phage on the binding between HCV E2 and human CD81 was detected. Result Thirty two phage clones were isolated after 3 rounds of biopanning from the peptide library, and 3 positive clones (C4,C13,C16) were confirmed by ELISA and DNA sequencing. The binding between clone C16 and HCV E2 could be inhibited by sera from immunized mice. The C16 clone could inhibit the binding between CD81 and HCV E2 in dose-dependent manner. Conclusion The amino acid sequences of the positive clones shared no homology with hCD81, and could be considered as the mimic peptides of hCD81. An identified peptide can competitively inhibit the binding between HCV E2 and CD81. These clones may have potential applications in the study of anti-HCV drug and vaccine development.