Production Of BNK19-Specific Monoclonal Antibody Of C-type Lectin Like BNK Protein Expressed On Chicken NK Cell

Natural killer(NK) cell is an important component of innate immune system of mammals and other vertebrate with a function of antineoplastic and antiviral infection. The foundation of NK cell is related to signals mediated by activating receptors or inhibiting receptors. Currently, chicken NK cell research has been paid more and more attention, but the lack of NK cell-specific surface marker hampers the advancement. Chicken BNK protein, encoded by b-nk gene, is known expressed on NK cells with a C-type lectin like structure. BNK is highlyglycosylatedⅡtransmembrane protein. It is most homologous to human NKRP-1 and it has a functional cytoplasmic ITIM. Therefore, BNK protein is a potential NK cell receptor.b-nk gene is located in major histocompatibility complex(MHC) core region and is closely linkaged with other MHC-B core genes. In this study, 6MHC haplotype chicken serial BW/G lines including B19 and B21 stored in National Resource Center for Poultry Laboratory Animal are used as experimental materials. The genetics polymorphism of b-nk genes of the 6 lines were compared, the result shows that all the b-nk genes identical to the corresponding standard haplotypes. It suggests that b-nk gene can be used to monitor genetics stability of MHC haplotype chicken and the lines could be used in the following research.B19 haplotype chicken is susceptible to several chicken diseases. In this study, BNK19(i.e. BNK protein of B19 chicken) is expressed by Escherichia coliand one monoclonal antibody strain of 7A10 specific to BNK19 was prepared. Western blotting showed that 7A10 could not detect the BNK21 protein produced by Escherichia coli. By use of truncating expression of prokaryotic BNK the epitopeof 7A10 was targeted between E149-S168.b-nk19 gene was amplified from total mRNA extracted B19 haplotype peripheral blood lymphocytes(PBL) and inserted to eukaryonexpression vector to obtain pIRES-BNK19 of recombinant plasmid. Followed by transfecting into CHO cell line, a stably expressed BNK19 cell line was obtained.7A10 can detect the BNK protein from PBL by Western Blot. 16.7% PBL can be stained by 7A10 by flow cytometry analysis. This study provides foundation for further study of BNK protein in chicken NK cell function.