筛选诱导肠道病毒71型特异性免疫应答的最短氨基酸基序

Screening specific minimum amino acid sequence triggering immunity to enterovirus 71

目的筛选肠道病毒71型(EV71)中和表位,确定诱导机体免疫应答的特定最短氨基酸基序,为合成肽疫苗研发提供理论依据。方法利用噬菌体随机十二肽库淘选EV71中和抗体特异性结合克隆并测序,推导出中和表位的关键序列。合成含有关键序列且N端乙酰化(AC)和C端连接血蓝蛋白(KLH)的系列肽段,免疫小鼠后收集血清,经Western blot、ELISA和中和试验验证肽段的免疫原性以及免疫后血清的中和活性。结果经过3轮淘选和克隆测序后,分析确定关键基序为KQEKDL。Western blot结果表明修饰后的含关键基序的系列肽段免疫小鼠获得的血清与EV71和VP1蛋白均有不同程度结合。ELISA结果表明仅含有最短关键基序的合成肽段(AC-KQEKDL-KLH)免疫后血清对其余3条肽段及EV71的反应最强。中和试验结果表明该肽段免疫后血清的中和效价也最高。结论利用噬菌体随机肽库技术成功筛选出EV71中和表位,确定KQEKDL关键基序可能是诱导机体免疫应答的特定最短氨基酸序列,为了解免疫应答机制、免疫原性评价及多肽疫苗的研发提供理论依据。

Objective To screen the neutralizing epitope of enterovirus 71(EV71)and determine the specific minimum amino acid sequence that triggers immunity for providing a theoretical basis for the development of synthetic peptide vaccines.Methods EV71 neutralizing antibody-specific binding clones were panned and sequenced using a phage display random 12-peptide library to obtain the key sequences of neutralizing epitopes.A series of peptides containing the key sequences with N-terminal acetylation(AC)and C-terminal linking to Keyhole limpet hemocyanin(KLH)were synthesized.Serum samples were collected after immunizing mice with the modified peptides.Then the immunogenicity of the peptides and the neutralizing activity of serum samples were analyzed by Western blot,ELISA and neutralization test.Results After three rounds of panning,cloning and sequencing,KQEKDL was identified as the key motif.The serum samples collected from the mice immunized with the modified series of peptides containing key motifs had different degrees of binding ability to EV71 and VP1 protein.The serum samples of mice immunized the synthetic peptide containing only the minimum key motif(AC-KQEKDL-KLH)had the strongest response to the other three peptides and EV71 and the highest neutralizing titer.Conclusions The EV71 neutralizing epitope was successfully screened using the phage display random peptide library.The key motif of KQEKDL might be the specific minimum amino acid sequence that triggered the immune system.This study provides a theoretical basis for better understanding the immune response mechanism,evaluating the immunogenicity of the antigens and further research and development of polypeptide vaccines.