噬菌体肽库的构建及抗狂犬病病毒肽的筛选

Construction of Random Peptide Library in Bacteriophage and Screening of Peptide Antirabies Virus

体外合成编码６肽的随机ＤＮＡ片段以及用于随机。ＤＮＡ片段扩增的１对ＰＣＲ引物，经ＰＣＲ扩增、ＢｇｌⅠ酶切扩增产物，得到编码６肽的随机ＤＮＡ克隆片段，将随机ＤＮＡ克隆片段与噬菌体表面表达载体（ＦＵＳＥ５）相连接，连接产物电转化ＭＣ１０６１宿主菌，经抗性和插入复活筛选，获得１．６×１０８个独立克隆。文库经ＰＣＲ扩增、斑点杂交、序列测定等综合鉴定，结果表明已成功构建了噬菌体６肽表面表达文库。用生物素化的抗狂犬病病毒单克隆抗体（９Ｅ６）同狂犬病病毒ａＧ株作用，再同包被亲和素的反应板作用，以此系统对噬菌体６肽随机文库进行３轮亲和筛选，用人抗狂犬病病毒抗体和抗噬菌体抗体对第３轮筛选扩增物进行特异性鉴定，结果表明，得到的扩增物均能与狂犬病病毒ａＧ株发生特异性结合。对２种鉴定系统均是阳性的噬菌体克隆进行序列测定，结果可分为４组核心序列，分别为ＴＰＰＲＰＰ（６Ｐ１－ＲＶ）、ＴＰＰＩＰＰ（６Ｐ２－ＲＶ）、ＩＫＰＰＰＰ（６Ｐ３－ＲＶ）、ＰＰＲＰＰＲ（６Ｐ４－ＲＶ）。将以上４组核心序列同蛋白数据库进行同源性比较，发现４组肽序列同已知的狂犬病病毒受体乙酰胆碱受体序列无同源性，提示为一组新的结合狂犬病病毒的短肽。４种混合噬菌体肽对狂犬?

Random oligonucleotides encoding hexapeptides and a pair of primers for amplifying the oligonucleotides were synthesized. The PCR products digested with BglⅠ were directly inserted to Phage-Vector (FUSE5), in which their ligatures were electroporated into insensitive bacterium of MC1061. 1.6×108 clones obtained through screening of tetracycline resistance and insertion reactivation. The random hexapeptide library was proved to be successfully constructed by PCR amplification, oligodeoxynucleotide hybridization, necleotide sequencing. In this study, the peptide library was panned with rabies virus (aG strain) in order to get phage binding rabies virus. The phages binding rabies virus were detected with ELISA of using human antibodies against rabies virus and rabbit antibodies against bacteriophage after three rounds of panning and the four consensus motif identified, that is TPPRPP (6P1RV), TPPIPP (6P2RV), IKPPPP(6P3RV) and PPRPPR(6P4RV) respectively, which bound rabies virus with the different affinities. The homology of the four motif were compared with protein sequence databank. The results showed that the four motif have no homology with sequence of acetylcholine receptor of rabies virus indicating there may be a new group of short peptides binding rabies virus. The compound of four peptides displayed on phage surface partially protected mice from challenge of rabies virus.