Molecular cloning, expression, and purification of pig interleukin-5.
Interleukin-5 (IL-5) is thought to be a key cytokine in allergic inflammation. Pig IL-5 was cloned, sequenced, and expressed to enable us to study of the biological role of IL-5 in pigs used in a model for allergen-induced late-phase reactions. These pigs were sensitized to proteins extracted from Ascaris suum, resulting in hypersensitivity to this antigen in both the skin and airways, and a slight blood eosinophilia. Peripheral blood mononuclear cells from antigen-sensitized pigs were isolated and polyclonally stimulated. Total RNA was extracted and reverse transcribed into cDNA. IL-5 primers based on the cow IL-5 cDNA sequence were used to obtain an initial polymerase chain reaction product. 3' rapid amplification of cDNA ends (3'RACE) and 5'RACE procedures were applied to identify the 3' and 5' ends, respectively. The full-length pig IL-5 cDNA is 405 base pairs long. Mature pig IL-5 was expressed in Escherichia coli with a His-tag for purification. The IL-5 protein is 115 amino acids long, has an estimated molecular weight of 14 000 M(r) and forms a biologically active homodimer of 28 000 M(r). Pig IL-5 shows 65% amino acid identity to the human IL-5 sequence and 90, 88, 83, 62, and 61% identity to the cow, sheep, horse, mouse, and rat counterparts.
Sylvin
H
H
Department of Laboratory Medicine, Division of Clinical Immunology, Karolinska Hospital, SE-17176 Stockholm, Sweden. helena.sylvin@labmed.ki.se
Matvienko
O
O
Leonchiks
A
A
Alving
K
K
van der Ploeg
I
I
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D017165:Ascaris suum / Q000276:immunology; D001249:Asthma / Q000276:immunology; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D019281:Dimerization; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D015848:Interleukin-5 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; D015848:Interleukin-5; D011993:Recombinant Fusion Proteins