小鼠TLR-2N末端基因的克隆表达和抗体制备

目的 克隆mTLR-2基因氨基端序列,并表达和纯化N端融合蛋白,制备抗mTLR-2 N末端的多克隆抗体.方法 采用PCR技术扩增编码mTLR-2N端1～153氨基酸的基因,并将其克隆到pET32A载体中,测序验证;用大肠杆菌表达融合蛋白,并以Probond树脂柱纯化;免疫家兔制备多克隆抗体,采用免疫组化、流式细胞术及间接ELISA检测抗体特异性及效价.结果 成功构建了融合表达载体pET-N,表达和纯化了相应的融合蛋白,所制备多克隆抗体可与pGEX-N表达的融合蛋白反应,并可结合表达TLR-2的RAW264.7及转染mTLR-2全长基因的CHO细胞.结论 获得了mTLR-2 N末端重组融合蛋白及其可与天然mTLR-2结合的多克隆抗体.

Cloning and expression of murine Toll-like receptor-2 N terminal and preparation of its antibody

Objective To prepare the recombinant murine Toll-like receptor-2 N-terminal(mTLR-2N) fusion protein and obtain anti-mTLR-2N polyclonal antibody.Methods The gene encoding 153 amino acids of mTLR-2N was amplified by PCR and cloned into pET32A vector with sequence verification.The recombinant fusion protein was expressed in E.coli and purified by Probond resin column.Rabbits were immunized with fusion protein to obtain the polyclonal anti-sera,and the antibodies were identified by enzyme-linked immunosorbent assay(ELISA) and immunohistochemistry.Results The recombinant fusion protein was efficiently expressed and purified.The polyclonal antibodies could bind to the fusion protein expressed in different vectors as the antigens in ELISA,and also bind with RAW264.7 cells expressing mTLR-2 and CHO cells transfected with full-length mTLR-2 gene.Conclusion The recombinant mTLR-2N fusion protein is obtained and the anti-mTLR-2N polyclonal antibody can recognize natural mTLR-2 on the cell surface.