人源单克隆抗-P免疫球蛋白基因可变区序列分析OA

Objective To identify the structure of the complementarity determining region(CDRs),the V(D)J rear-rangement and somatic hypermutational characteristics of the heavy and light chains of a red blood cell blood group-specific monoclonal antibody.Methods The hybridoma cell line secreting human IgM κ monoclonal anti-P antibody was used as the research object.Total RNA was extracted from cultured monoclonal cell line,and cDNA was obtained by reverse tran-scription PCR(RT-PCR)using random hexamers primers.It was then amplified and sequenced using primers specific for variable regions of the immunoglobulin heavy and light chains encoding the anti-P antibody.The sequences were aligned a-gainst the NCBI database using online Immunoglobulin BLAST(Ig-BLAST)tool.Results The study determined the struc-ture of the CDRs and framework regions(FRs)of the variable regions of human monoclonal anti-P immunoglobulin,as well as the characteristics of V(D)J rearrangement.Moreover,the closest VH,VD,and VJ germline alleles for the heavy chain and VL and VJ germline alleles for the light chain were also identified.The IgH gene rearrangment pattern of the monoclonal anti-P was IGHV6-1∗01—IGHD5-18∗02—IGHJ4∗02 and IgL gene was IGκV1-12∗01—IGκJ3∗01.Nine base mutations occurred within the germline gene IGHV6-1∗01 in variable region of heavy chain,whereas 5 base mutations were found in the germline gene IGκV1-12∗01 in variable region of light chain,respectively.Conclusion This study characterized the CDR structure in monoclonal antibody cell line targeting the high-frequency red blood cell P antigen,and provided a founda-tion for the construction of recombinant antibody expressing plasmids and transfomation of the immunoglobulin type.