Rapid cloning of HLA class I cDNAs by locus specific PCR.
The human major histocompatibility complex (MHC) class I loci, human leukocyte antigen (HLA)-A, -B, -C, encode highly polymorphic molecules that mediate immune recognition of infectious pathogens and can initiate the rapid rejection of transplanted tissue. Cloning of HLA class I alleles is complicated by polymorphism as well as interlocus homology. Here, HLA class I cDNAs are amplified by PCR using one common primer with one of three locus specific primers whose 3' ends map to conserved, locus specific nucleotides. Using these primers, HLA-A, -B, and -C alleles were cloned from a number of cell lines and two different HLA-B alleles were cloned from a single, heterozygous cell line. The amplified products encode the entire extracellular portion of the class I molecules. An amplified HLA-A allele was cloned into an expression vector and the protein product was detected on the surface of a transfected cell. A premature termination codon was engineered into the HLA-A allele by site directed mutagenesis and the soluble protein product was detected in the culture medium of transfected cells. Therefore, these primers can be used to rapidly clone, alter, and express HLA class I molecules. This method may expedite the generation of reagents for testing the antigen specificity of antibodies, natural killer cells, or T cells.
Johnson
D R
DR
Department of Pathology, 454 Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT, USA. david.johnson@yale.edu
Biedermann
B C
BC
Mook-Kanamori
B
B
D000483:Alleles; D001483:Base Sequence; D002462:Cell Membrane / Q000276:immunology; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D005069:Evaluation Studies as Topic; D005805:Genes, MHC Class I; D006680:HLA Antigens / Q000235:genetics*; D015234:HLA-A Antigens / Q000235:genetics; D015235:HLA-B Antigens / Q000235:genetics; D015236:HLA-C Antigens / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012995:Solubility; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D006680:HLA Antigens; D015234:HLA-A Antigens; D015235:HLA-B Antigens; D015236:HLA-C Antigens