抗小鼠PGRP-L单表位多克隆抗体的制备

Preparation of anti-mouse PGRP-L single-epitope polyclonal antibody

目的制备抗小鼠长型肽聚糖识别蛋白(mPGRP-L)单表位多克隆抗体。方法应用生物信息学技术预测小鼠mPGRP-L分子的B细胞优势表位,人工合成抗原肽,采用MBS法将其与钥孔血蓝蛋白(KLH)偶联,免疫家兔获取mPGRP-L抗血清,采用HiTrap proteinG柱和抗原肽亲和层析柱纯化抗体,以ELISA和Western blotting进行鉴定。结果确定了位于mPGRP-L分子N端第85～104位残基的1个B细胞优势表位NH2-(C)DPHSLSPELQALISEVAQHD-COOH,合成短肽并制备KLH-肽偶联物,免疫家兔得到的抗血清效价达1∶256000。分别以HiTrapproteinG柱和抗原肽柱亲和层析纯化获得兔抗mPGRP-L单表位多克隆抗体mPGRP-Ln1和mPGRP-Ln2。纯化抗体能与重组蛋白pET-mPGRP-Ln结合,经Western blotting分析,在相对分子质量约29000处可见清晰的反应条带。结论获得抗mPGRP-L单表位多克隆抗体,为mPGRP-L分子的研究提供了工具。

Objective To prepare a single-epitope polyclonal antibody against mouse long peptidoglycan recognition protein （mPGRP：L）. Methods B cell dominant epitopes of mPGRP-L predicted by bioinformatics were synthesized, and the immunogen was prepared by conjugation of the synthetic peptide and the carrier protein key-hole limpet hemocyanin （KLH） by MBS method. The single-epitope polyclonal antibody was obtained by immunizing rabbits with the KLH-peptide conjugate, purified by SPG affinity columns or antigenic peptide affinity columns, and identified by ELISA and Western blotting. Results and Conclusion A dominant epitope in N-terminal region of mPGRP-L, with amino acid sequence of NH2- （C）DPHSLSPELQALISEVAQHD-COOH, was chosen and synthesized. The titer of anti-serum of the rabbits immunized with the KLH-peptide conjugate was 1：256 000. The polyclonal antibody purified with SPG affinity columns and antigenic peptide affinity columns were named as mPGRP-Lnl and mPGRP-Ln2, respectively. Western blotting demonstrated that the antibody mPGRP-Lnl could recognize the recombined N-terminal fragment of mPGRP-Ln with a clear band at relative molecular mass of about 29 000, suggesting the successful preparation of the single-epitope polyclonal antibody against the N-terminal region ofmPGRP-L.