Purification Of HCLEC-2 Recombinant Protein In Engineering Bacteria And Preparation And Identification Of Its Antibody

hCLEC-2(human C-type lectin-like receptor-2) is a newly identified typeâ…¡transmembrane receptor protein.It is found to be closely associated with virus infection,platelet aggregation,tumor metastasis and signal transduction.To study the biological function of hCLEC-2 and signaling pathways it is involved in,we intended to prepare an antibody specific to the extracellular domain of hCLEC-2.Then,a pET23b-CRD recombinant plasmid was constructed and transformed into BL21 for protein expression.After the induction of IPTG,hCLEC-2-CRD-His was found to be expressed in the inclusion body.The fusion protein in inclusion body was dissolved in 6M guanidine,purified using Ni-agarose,refolded and dialyzed against PBS.The purified hCLEC-2-CRD-His was used as an antigen to prepare polyclonal antiserum in rabbits,which was subjected to affinity purification with Protein G Sepharose.The specificity of anti-CRD antibody was identified by Western Blot analysis of the ectopic expressed GFP-hCLEC-2 and GFP-CRD in comparison with that of GFP antibody.Using this specific antibody,we found that the endogenous hCLEC-2 was down-regulated in human monocyte THP-1 cells treated with PMA and IL-4.This preliminary result suggests a correlation between the expression of hCLEC-2 and the differentiation of monocyte.Collectively,the studies here provide a favorable tool for further investigation of hCLEC-2 associated biological functions.