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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Food Safety and Enteric Pathogens Research » Research » Publications at this Location » Publication #126092
Title: DEVELOPMENT OF A BACULOVIRUS EXPRESSION SYSTEM FOR SOLUBLE PORCINE TUMOR NECROSIS FACTOR RECEPTOR TYPE I AND SOLUBLE PORCINE TUMOR NECROSIS FACTOR RECEPTOR TYPE I-IGG FUSION PROTEIN

Author
item MWANGI, SIMON - NATL ANIM DIS CTR
item STABEL, THOMAS
item KEHRLI JR, M - PFIZER INC

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/20/2002
Publication Date: N/A
Citation: N/A

Interpretive Summary: In addition to the economic impact of nontyphoid salmonellosis on the human population, it is a major economic disease of swine and cattle resulting in millions of dollars in lost income to both industries. A factor called tumor necrosis factor alpha (TNF alpha), produced during swine salmonellosis, may be crucial to disease resistance but may also contribute to the disease process and have lingering effects on performance. There is compelling evidence in the literature that recombinant soluble TNF receptor 1 (rsTNF-R1) can effectively block TNF alpha in disease circumstances, and reduce (or abolish) tissue damage and adverse clinical signs. It is our belief that if natural and safe inhibitors of TNF alpha were available, they could be used to effectively block the excessive and long-term negative effects of TNF alpha during various diseases of livestock, and hence, improve performance. Scientists at the National Animal Disease Center, Ames, IA, developed a cost effective method for the construction and production of large-scale amounts of biologically active rsTNF-R1 and rsTNF-R1 fused to a carrier protein, the Fc region of porcine IgG1. The fusion protein (rsTNF-R1-IgG) was approximately 10 times more effective at inhibiting TNF alpha activity than receptor alone. Results of this study prove that it is possible to design essentially natural inhibitors of porcine TNF alpha. These new products will have clinical application for food animals. Eventual beneficiaries are the American consumer since new intervention strategies for salmonellosis will allow a continued supply of inexpensive, wholesome pork and pork products.

Technical Abstract: Tumor necrosis factor alpha (TNF alpha)is a key mediator of inflammatory responses and gram-negative bacterial sepsis; however, the role that TNF alpha plays in the pathogenesis of Salmonella enterica species in swine has not yet been elucidated. To facilitate studies on the role of TNF alpha in septicemia and tissue damage associated with Salmonella enterica species infections in pigs, recombinant soluble porcine TNF receptor type I (rspTNF-RI) and soluble TNF receptor type I fused to the Fc region of porcine IgG1 (rspTNF-RI-IgG) were expressed in insect cells using a baculovirus expression system. The proteins were secreted into the cell culture media and purified by anti-soluble porcine TNF-RI antibody and protein G affinity chromatography, respectively. The yield of protein using this method was approximately 1.5 mg rspTNF-RI and 4 mg rspTNF-RI-IgG/L of cell culture medium. In in vitro assays, rspTNF- RI-IgG was approximately 10.3 fold (9.67 x 10**-1 vs. 10 picomoles/ml) more effective than rspTNF-RI at completely inhibiting the cytotoxic activity of 500 U of recombinant porcine TNF alpha on 3 x 10**4 WEHI 164 murine fibrosarcoma, clone 13, cells. Compared to previously described methods, this method yields significantly more biologically active rspTNF-RI.