Cloning and characterization of the bovine interleukin-2-gene

Details Cloning and characterization of the bovine interleukin-2-gene Cloning and characterization of the bovine interleukin-2-gene

C - Chemistry, Metallurgy

12

N

530/13, 167/103.

C12N 15/26 (2006.01) A61K 38/20 (2006.01) C07K 14/55 (2006.01) C12N 1/19 (2006.01) C12N 1/21 (2006.01) C12N 5/10 (2006.01) C12N 15/63 (2006.01) C12N 15/73 (2006.01)

Patent

CA 1341220

A chemically-synthesized oligonucleotide composing a portion of the nucleotide sequence of the human IL-2 is employed as a probe to isolate the gene coding for human IL-2. The human IL-2 gene is selected from a cDNA library prepared from RNA produced by mitogen-stimulated Jurkat cells. Double-stranded eDNA is prepared from polyadenylated RNA extracted from bovine cells thought to produce interleukin-2. Such cDNA is inserted within a plasmid vector and the recombinant plasmid employed to transform hosts. Plasmid DNA, prepared from pools of the transformed hosts, is hybridized with a probe composed of a large portion of the coding sequence of the human IL-2 gene. Pools of host cells that provide a positive signal to the human eDNA probe are identified, subdivided, and rescreened until a single positive colony is identified. Bovine plasmid cDNA is prepared from this colony, and the bIL-2 gene is sequenced. The plasmid DNA is employed to express recombinant bovine IL-2 in yeast and bacterial expression systems, with the expressed bovine IL-2 purified to homogeneity by one or more reverse phase high-performance liquid chrom atography procedures.

516032

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