Preparation of monoclonal antibody of human C-type lectin receptors dectin-1

Objective: To clone and prokaryoticly express the extracellular region of human C-type lectin receptor dectin-1 and prepare monoclonal antibodies of human dectin-1. Methods and Results: The full-length human dectin-1 mRNA was cloned from peripheral blood monocytes by using RT-PCR and inserted into the pCDNA3.0(+) vector. The extracellular region of dectin-1 [dectin-1 (202 bp-744 bp)] was cloned and inserted into pET28a (+) vector to get pET28a (+)-dectin-1 (202 bp-744 bp) vector. The recombinant pET28a-dectin-1 (202 bp-744 bp) vector was transformed into E. coli BL21 (DE3). The expression of objective protein was high when induced with IPTG. The objective protein was purified with Ni-NTA affinity Chromatograph and was used to immunize BALB/c mice. The mice which generated higher titer of antibody in the serum were sacrificed. The splenocytes were separated and fused with SP2/0 cells to generate hybridoma cells. Fifteen strains of hybridoma cells were thus obtained. After further selection and cloning, 5 strains of monoclonal hybridoma cells secreting monoclonal antibodies against dectin-1 were established. BALB/c mice was intraperitoneally injected into hybridoma cells to produce ascites. The dectin-1 monoclonal antibody was purified by standard protocol of octanoic acid-ammonium sulfate precipitation, which could specifically combine to dectin-1, no matter whether they were human dectin-1 or mouse dectin-1, when used in Western blotting. Conclusion: The specific monoclonal antibodies against dectin-1 were successfully obtained.