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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Biosciences & Biotechnology Laboratory » Research » Publications at this Location » Publication #360032

Research Project: Non-antibiotic Strategies to Control Enteric Diseases of Poultry

Location: Animal Biosciences & Biotechnology Laboratory

Title: Expression and characterization of chicken perforin and granzyme a

item Li, Charles
item SUN, ZHIFENG - US Department Of Agriculture (USDA)
item KIM, WOOHYUN - US Department Of Agriculture (USDA)
item Lillehoj, Hyun
item ZHAO, HONGYAN - US Department Of Agriculture (USDA)
item LU, MINGMIN - US Department Of Agriculture (USDA)

Submitted to: American Association of Avian Pathologists
Publication Type: Abstract Only
Publication Acceptance Date: 2/17/2019
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Perforin and granzymes are pore forming cytolytic proteins and serine proteases, respectively, found in the granules of cytotoxic T lymphocytes (CTLs) and Natural Killer (NK) cells, and are important players in immune responses. Upon degranulation, perforin binds to the target cell's plasma membrance, and form pores on the target cell, allowing for the passive release of granzymes from endolysosomal vesicles into the target cells. Granzyme B does not seem to exist in chickens. Granzyme A is one of the most biologically important granzymes which induce programmed cell death (apoptosis) in the target cells, thus eliminating cells that have become cancerous or are infected with viral or bacterial pathogens. Perforin-mediated cytotoxicity is crucial for control of intracellular pathogens. In this study, chicken perforin (ckPRF1) and granzyme A (ckGZMA) proteins were expressed and characterized. A 2478-bp extracellular region of ckPRF1 gene was cloned in pET28a(+) vector and expressed in BL21-AI™ E. coli. The recombinant ckPRF1 proteins were around 46kDa, 25kDa and 16 kDa, and cross-reacted with monoclonal antibody (mAb) specific for human PRF1 (Clone F-1). A 711 bp extracellular region of ckGZMA gene was cloned in pRSET-C expression vector. The recombinant plasmids were transformed into E. coli strain BL21 Star (DE3) (pLysS) cells. The expressed ckGZMA protein was around 37 kDa, and cross-reacted with a mAb specific for human GZMA (Clone 3G8.5). These proteins will be used for mAb development which will be helpful for the detection of these proteins in biological samples. Availability of these antibodies for PRF1 and other CD molecules will facilitate the commercialization of poultry immune reagents, greatly benefiting the avian immunology community and promote poultry health and production, where it will have a significant global impact in multiple sectors (i.e. agriculture and human healthcare).