摘要
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．coliB121（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21．80kDa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1：25600，Westernblot分析显示抗体能特异性结合人TREM一1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。
Objective To study the expression of a prokaryotic expression vector for human trigge- ring 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.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2011年第9期1438-1440,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（30972779）
广东省科技计划资助项目（20088030301022）
