[Preparation and characterization of polyclonal antibodies against haplotype chicken natural killer cell receptor B-NK19 and B-NK21].

OBJECTIVE:To express natural killer (NK) cell inhibitory receptor gene B-NK of B19 and B21 haplotype chicken, which show different susceptible to various avian diseases, using prokaryotic expression system, and to prepare B-NK19 and B-NK21-specific rabbit polyclonal antibodies. METHODS:B-NK genes of B19 (named as B-NK19) and B21 (named as B-NK21) chicken were amplified by reverse transcription PCR from peripheral blood lymphocytes of one B19 or B21 chicken, respectively, and then inserted into an expression vector of pET-30a. The recombinant plasmids of pET-B-NK19 and pET-B-NK21 were transformed into E.coli (DE3) and induced to express, respectively. Both His-tagged foreign proteins of B-NK19 and B-NK21 were seperated by SDS-PAGE and immunized to New Zealand rabbits to prepare polyclonal antibodies. The MHC-specificities of the sera were analyzed by Western blotting. RESULTS:The chicken NK cell inhibitory receptor gene B-NK from MHC haplotypes B19 and B21 were successfully amplified, cloned and expressed in E.coli after induction with 1.0 mmol/mL IPTG for 4 h at 37°C, respectively. Western blotting revealed that the prepared rabbit serum antibodies positively reacted to the expressed foreign proteins. CONCLUSION:Chicken NK cell inhibitory receptor proteins B-NK of B19 and B21 haplotypes could immunologically cross-react and their polyclonal antibodies are partially specific to B complex.