HLA-A2单抗轻链可变区基因的克隆及其序列分析

CHARACTERIZATION OF MOLECULAR STRUCTURE FOR IMMUNOGLOBULIN VK REGIONS OF MURINE MONOCLONAL ANTIBODIES AGAINST HLA-A2

以HLA—A2单抗mRNA为模板,设计了3对特异性引物,采用PCR技术,扩增和克隆出单抗轻链可变区基因并应用双脱氧链式终止法及计算机基因文库测定和分析了其氨基酸和核苷酸序列。结果证明,本组单抗轻链由不同的基因家族编码。

We have characterized and determined the V region gene sequences of a series of mouse monoclonal anti-HLA-A2 kappa chains. Molecular and structural studies of antibody V regions have provided information on Ag binding and Id expression and construction of genetic engineering antibody. Total cellular RNA was isolated from hybridoma cell lines producing five monoclonal antibodies against HLA-A2. By reverse transcription the cDNAs were synthesized and used as templates for PCR. The PCR products were digested with EcoR I and Hind I and ligated to pUC19 or pUC18 predigested with the same restriction endonucleases. We designed specific primers for the amplification VK genes. In the primers restriction endonucleases EcoR I and Hind I site were incorporated for forced cloning. By screening and identification several recombinants that had been inserted with the target fragments were obtained. The recombinants were sequenced with Sanger's method. It was confirmed by computer-assisted comparative sequence analysis that we have successfully cloned the full-length and potentially functional VK genes of the monoclonal antibodies against HLA-A2.