Recent Progress

Narrative report:  The initial step in our research was to clone the full-length yellow perch growth hormone (YPGH) cDNA. This was accomplished early in the first year of funding.  Using PCR, a cDNA fragment was isolated and the full-length clone was obtained using 5'/3' RACE (rapid amplification of cDNA ends). The resulting full-length clone is 935 bp with an open reading frame of 708 bp presumably coding for a 204 amino acid protein. The GenBank Accession number for this clone is AY007303.

The major goal of this research is to develop an antibody toYPGH that can be used to detect GH levels in yellow perch with some type of immunoassay. To do this a recombinant GH antigen is needed.  To create recombinant GH the open reading frame of YPGH was cloned into a vector that facilitates the translation and expression of the protein. A number of systems were tried including the pMAL (New England Biolabs), pET (Novagen), IMPACT (New England Biolabs), and pBAD (Invitrogen) protein expression systems.  After Western analysis it was determined that the pBAD system produced sufficient amounts of YPGH for immunization (figure 1, lane A).  The purified pBAD fusion protein was used to immunize rabbits.  Following a series of booster injections, immune serum was collected after 52 days. Immunoglobulins were purified and used in Western analysis to determine their recognition of native YPGH (figure 1, lane B).  To analyze native GH, whole pituitary samples were used because of the difficulty of purifying GH from pituitaries and the fact that almost 50% of the contents of the pituitary is GH.  The single band observed in lane B appeared to be native YPGH from the pituitary based on its size of ~21kDa.  To verify that the antibody was recognizing GH, pure recombinant sea bream GH (GroPep Limited) was used to test the antibody.  Sea bream GH was chosen because of the close phylogenetic relationship and corresponding high protein sequence homology (>90% to yellow perch GH).  As seen in figure 2, the GH-pBAD antibody did recognize sea bream GH (lane A).  Yellow perch (lanes B & C) and brook trout (lane D) pituitary samples were also analyzed.  The GH-pBAD antibody did not recognize brook trout GH as well as the other species.  This is expected due to the distant phylogenetic relationship of the species and is evidence that the antibody is relatively specific for perciform GHs.
In collaboration with Dr. Jeff Malison (Aquaculture Program at the University of Wisconsin-Madison) we are currently assaying yellow perch pituitaries seasonally and under different environmental conditions using Western analysis to observe changes in GH levels.  Observing changes in GH hormones levels will give researchers and aquaculturists a better understanding of the growth process in yellow perch and the factors that control growth.
While western analysis will suffice as a temporary measurement tool, the GH-pBAD antibody should be used for the development of an immunoassay that would allow for higher throughput and accurate quantitation of many samples.  To develop a precise YPGH standard for such an immunoassay, a pure recombinant protein is required and this is currently being pursued.