MOLICULAR CLONING EXPRESSION AND PURIFICATION OF PORCINE IL1-A AND B

THE LONG-TERM (PHASEII) PURPOSE OF THIS RESEARCH IS TO CLONEAND EXPRESS THE COMPLEMENTARY DNAS (CDNA) ENCODING PORCINE INTERLEUKIN 1 (IL1)-A AND-B, AND PURIFY BOTH CYTOKINES TO HOMOGENEITY. ALTHOUGH POTENTIAL CLINICAL APPLICATIONS FOR THE CYTOKINES ARE INDICATED, THESE ARE NOT INCLUDED IN THIS PROPOSAL NOR IS SUPPORT FOR CLINICAL STUDIES REQUESTED. IN PHASE I, SWINE ALVEOLAR MACROPHAGES ELICITED IN RESPONSE TO INFECTION WITH ASCARIS SUUM WILL BE CULTURED INTHE PRES- ENCE OF A STIMULATORY DOSE OF LIPOPOLYSACCHARIDE OR CONCANA-VALIN A AND MRNA WILL BE ISOLATED. NORTHERN BLOT ANALYSES WILL BE PERFORMED TO DETERMINE CONDITIONS UNDER WHICH POR- CINE IL1 MRNAS ARE OPTIMALLY PRODUCED. LARGE-SCALE MRNA PREPARATIONS WILL BE MADE. CDNA WILL BE SYNTHESIZED FROM THE MRNA, APPROPRIATELY MODIFIED FOR CONSTRUCTION OF A LIBRARY IN \GT10, AND USED TO INFECT A SUSCEP- TIBLE STRAIN OF E. COLI. INDEPENDENT PLAQUES WILL BE SCREENBY HYBRIDIZATION WITH HUMAN OR BOVINE IL1-A AND -B PROBES. CDNA INSERTS FROM CLONES DISPLAYING MAXIMAL HYBRIDIZATION WITH EACH PROBE WILL BE ISOLATED AND SUBCLONED. SEQUENCING EXPRESSION AND PURIFICATION WILL BE CONDUCTED IN PHASE II.