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

Abstract

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. Key words: TREM-1 ; Prokaryotic expression vector; Sepsis; Polyclonal antibody