人髓样细胞触发受体-1原核表达载体的构建和表达及多克隆抗体的制备

目的 构建人髓样细胞触发受体-1(TREM-1)原核表达载体使其表达并制备多克隆抗体。方法 采用特异性引物扩增人TREM-1胞外段cDNA,经酶切、连接、构建到原核表达载体pET28a(+),将构建的重组表达质粒转化入E.coli BL21( DE3)菌株,采用IPTG诱导表达、Ni-NTA 柱亲和层析纯化目的蛋白、SDS-PAGE分析蛋白纯度,将纯化的目的蛋白免疫家兔制备多克隆抗体,并对其进行纯化及鉴定。结果 序列测定证实构建的重组表达载体pET28a(+)-TREM-1含有人TREM-1编码序列,其序列分析与Genbank中公布序列对比一致,质粒在E.coli中诱导表达相对分子质量(Mr)为21.80 kDa的目的蛋白,SDS-PAGE分析表明纯化后的目的蛋白达到电泳纯,双向琼脂扩散法检测抗体效价为1:16,ELISA法检测抗体效价为1:25 600,Western blot分析显示抗体能特性结合人TREM-1重组蛋白。结论 成功构建高表达重组人TREM-1蛋白的原核表达载体及制备高效价兔抗人TREM-1多克隆抗体。

Construction and expression of a prokaryotic expression vector for human TREM-1 and preparation of a polyclonal antibody

Objective To study the expression of a prokaryotic expression vector for human triggering receptor expressed on myeloid cell ( TREM-1 ) and preparation of a polyclonal antibody. Methods Human TREM-1 gene was amplified with specific primers. The polymerase chain reaction (PCR) product was digested with enzymes and then linked into the recombinant prokaryotic expression vector pET28a ( + ). The expression vector was transformed into E. coli BL21 (DE3) and induced with IPTG to express human TREM-1 fusion protein that was then purified with Ni-NTA purification system and analyzed with SDS-PAGE. The purified fusion protein was used to prepare polyclonal antibody in rabbits. Results The sequence analysis confirmed that this recombinant expression vector contained human TREM-1 coding sequence that was identical with the published sequence in Genbank. The plasmid expressed a 21.80 kDa protein. After purification with Ni-NTA purification system, the resulted protein was analytically pure according to the analysis with SDS-PAGE. The titer of the antiserum was 1 : 16 and 1 : 25 600 detected by double diffusion test and ELISA, respectively. Western blotting analysis demonstrated that the anti-TREM-1 antibody bound specifically human TREM-1 recombinant protein. Conclusion The prokaryotic expression vector and the polyclonal antibody against human TREM-1 were prepared successfully.