Mutagenesis and cloning of human CD4 gene by using polymerase chain reaction

CD4 molecule is an important differentiation antigen of T lymphocyte and has been identified as the receptor for HIV. We have designed and chemically synthesized two primers, and successfully amplified a gene fragment encoding the N-terminal two domains of human CD4 protein. EcoR I and Hind I recognition sites and the initiation and termination codons were incorporated into the 5' and 3' termini of this gene through polymerase chain reaction. The CD4 gene fragment was digested with EcoR I and Hind I and inserted into pUC19 plasmid. The recombinant clones were confirmed by polymerase chain reaction and restriction endonuclease cleavages and one of the correct clone was named pT403. The CD4 gene fragment in pT403 was sequenced by dideoxynucleotid termination method and it was revealed that the sequence of the cloned gene fragment was identical to the published CD4 cDNA sequence.