CLONING AND SPECIFICATION OF HUMAN INTERLEUKIN-GENE

摘要

Double-stranded cDNA is prepared from polyadenylated RNA extracted from activated human peripheral blood adherent mononuclear cells. The cDNA is inserted within a plasmid vector and then the recombinant plasmid employed to transform an appropriate host. Transformed hosts are identified and grouped into pools. Plasmid DNA prepared from these pools is hybridized with total mRNA from human peripheral blood cells. Pools of host cells that provide a positive signal from the hybrid section method are subdivided and the procedure repeated until a single positive colony is isolated. Plasmid DNA is prepared from this colony and radiolabeled for use as a probe to rescreen the cDNA library for a larger clone. Also a radiolabeled, synthetic oligonucleotide probe corresponding to a portion of the initially isolated clone is used to further screen the cDNA library for additional clones. Portions of the additional clones are then employed as labeled probes to further screen the cDNA library. By this process, a single clone spanning the entire open reading frame of the IL-1 gene was isolated as well as several other clones composing a portion of the gene. The entire open reading frame and the coding region of the IL-1 gene was inserted into expression vectors for expression of functional IL-1.