Cloning, Prokaryotic Expression And Purification Of The Conservation Area Polypeptide Containing CDR1 From Mouse TREM-1

Background and objective:Sepsis with high mortality rates makes damage to the people's health, and a lot of researchs about its mechanism have been done. Now it is known that sepsis arises from a cascade of releasing of pro-inflammatary cytokine after monocytes/macrophages activated by lipopolysaccharide(LPS). LPS-binding protein(LBP) can bind LPS and then transfer it to the cellular membrane. The signalling conduction is dependent on the presence of the Toll-like receptor 4 (TLR4), secreting protein MD-2 and membrane bound CD14 which can be a complex including LPS. TLR4 has a short-tail in the cytoplasm which can effect MyD88 and then lead to nuclear translocation of nuclear factor-kB (NF-kB) and ultimately to activate of cytokine gene promoters, at last the signal amplified. Triggering receptors expressed on myeloid cells-1(TREM-1) is recently discovered another important receptor which can amplify the inflammatory and maybe is one of the important target molecules to treat sepsis.TREM-1 is selectively expressed on blood neutrophils and CD14+ monocytes/ macrophages, and there are three antibody-equivalent complementarity determining region(CDR) on the surface of this receptor.TREM-1 have two forms in organism: membrane bound form and soluble form. Soluble TREM-1(sTREM-1) is the extracellular domain of the membrane bound form. Membrane bound TREM-1 can be activated upon stimulation with LPS and its nature ligand ligation. Activation of TREM-1 on monocytes up- regulates expression of the proinflammatory cytokines such as TNF-α, IL-6,IL-1βand GM-CSF and drives release of myeloperoxidase and robust production of proinflammatory chemokines such as monocyte chemokine protein-1 (MCP-1) and IL-8. In polymorphonuclear neutrophil (PMN), the engagement of this receptor on PMN can induce phagocytosis, respiratory burst and degranulation, while inhibiting production of IL-10, an anti-inflammatory cytokine. But sTREM-1 can inhibit production of proinflammatory cytokines and protect sepsis animal model to avoid high-reactivity and death to LPS. Otherwise, the amino acid residues closed to CDR1 are also high conservative and the function of this area hasn't been reported. So in our researchs, we have cloned and expressed the conservation area containing CDR1 of mouse TREM-1 and gained the fusion protein by affinity chromatograph and renaturation for investigating the function of this area of mouse TREM-1.Methods:The genomic DNA was extracted from the normal muscle tissues of a mature healthy Kunming mouse. The whole coding sequence of the conservation area containing CDR1 of mouse TREM-1 was initially obtained by PCR using designed primers.The PET-30a(+)-CDR1 prokaryotic vector was constructed by merging the fragment using gene engineering methods. The expression of the conservation area gene containing CDR1 of mouse TREM-1 in E. coli BL21(DE3)plysS was in form of fusion protein and induced by IPTG. The expression of this polypeptide was detected by Tricine-SDS-PAGE and Western blot and purified by affinity chromatograph. After renaturation, we get the fusion protein.Results:1.After homology analysis of TREM-1 amino-acid residues, it has been found that near CDR1, 31-56 amino-acid residues was high conservative. Contrasting the cDNA sequences, we found that the DNA sequences of the conservation area polypeptide containing CDR1 from mouse TREM-1 was in a exon, so the whole coding sequence could be amplified from the mouse genome DNA and the product's length would be 138 base pairs.2. The whole coding sequence was obtained by PCR and its length is 176bp because the primers was designed including restriction incision enzyme site and prokaryotic termination codon,etc.3. Both the PCR produce and plasmid were bouble digested by SacⅠand HindⅢand connected. The connective product was transformed into the E.coli DH5α. Then the plasmid was extracted and sequenced, and the result confirmed that the expressing plasmid has been constructed exactly.4. The recombination plasmid was transformed into the competence E.coli. BL21(DE3) plysS. Transformant colonies were selected and induced by IPTG to express the target fusion protein mostly in soluble form.About 12kD relative molecular weight proteins were detected by SDS-PAGE and it was found that the protein had high affinity to mouse Anti-His monoclone antibody by Western-blot.5. The fusion protein was isolated from the bacterias which have been induced by IPTG for 7 hours and purified by affinity chromatography using Ni-NTA agarose of Qiagen. After renaturation using the dialysis liquid containing GSH/GSSH, we got the fusion protein.Conclusions:1. The prokaryotic expression vector which can express the conservation area gene containing CDR1 of mouse TREM-1 was successfully constructed.2. We firstly found that the conservation area polypeptide could expressed by E.coli BL21(DE3)plysS at high level and mostly in soluble form.3. The fusion protein can be purified by Ni-NTA agarose. This results would contribute to further studies of the function of this conservation area polypeptide.