Performing Department
(N/A)
Non Technical Summary
The US dairy industry is dependent on having healthy dairy cows with high milk production and efficient reproduction. Unfortunately, dairy cattle can have a number of health and production problems that are not addressed by currently available methods and products. For example, new pharmaceutical products to improve dairy cattle health are surprisingly uncommon. Scienss Biotechnology, LLC (SB) has the technology, innovation, and flexibility to respond to this unique opportunity. This SBIR Phase I proposes new solutions to improve health and productivity of dairy cattle by addressing the core origins of most metabolic and disease problems that occur in lactating dairy cows. These core origins are the metabolic, hormonal, and health changes that occur near calving.The time period 21 days before and 21 days after calving are when 75% of health problems occur in dairy cows and is called the transition period. Two of the key problems that occur during this time will be addressed in the products that will produced during this research. The first issue is that not enough energy is consumed to meet the energy demands of the high milk production during early lactation, called negative energy balance. The second issue is that the large increase in feed consumption that accompanies early lactation can cause problems with the digestive tract, termed "leaky gut" and this can lead to systemic inflammation.SB will address these two problems and take advantage of the associated commercial opportunities by developing proprietary recombinant proteins designed to stimulate appetite and gastrointestinal (GI) tract health in transition dairy cows. Long-acting glucagon-like peptide-2 (GLP-2) will be designed to optimize function of GI tract and thereby reduce systemic inflammation in transitional dairy cows. Furthermore, a long-acting recombinant ghrelin protein will be designed to stimulate appetite and reduce negative energy balance in periparturient dairy cows. Thus, the long-term goals of this project are to produce, validate, and commercialize recombinant GLP-2, ghrelin, and other proteins to improve health and function of cattle, thereby increasing economic viability, sustainability, and efficiency of US dairy farms. Three specific objectives are proposed:Specific Objective 1: Express long-acting recombinant GLP-2 and ghrelin in 293F cells and evaluate immunological activity by ELISA.Specific Objective 2: Determine in vitro activity of native and recombinant GLP-2 and ghrelin using specific bioassays.Specific Objective 3: Determine in vivo biological activity and half-life for recombinant GLP-2 and ghrelin.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
This SBIR Phase I proposes new solutions to improve health and productivity of dairy cattle by addressing the core origins of most metabolic and disease problems that occur in lactating dairy cows, the transitional, periparturient period and associated negative energy balance (NEB) and systemic inflammation.Three major goals are proposed:Major Goal 1: Express long-acting recombinant GLP-2 and ghrelin in 293F cells and evaluate immunological activity by ELISA.Major Goal 2: Determine in vitro activity of native and recombinant GLP-2 and ghrelin using specific bioassays.Major Goal 3: Determine in vivo biological activity and half-life for recombinant GLP-2 and ghrelin.
Project Methods
For Specific Objective 1, NS-293 Free-style cells will be used to produce the recombinant proteins in suspension cultures. The transfected cells will be incubated in CD DHO medium supplemented with 8 mM L-GlutaMax in 125 mL shaker flasks (Corning). The NS-293 cells are adapted for suspension culture and designed to facilitate high protein production. The full-length sequences will be synthesized commercially through ATUM, and cloned into pcDNA3.1/ V5-His-TOPO mammalian expression plasmid. The sequence reading frame will be verified and confirmed by automated DNA sequencing. Expression vectors, with the verified sequence, will be expanded and purified for mammalian cell transformation. Transient transfection will be performed to determine if the protein is being expressed. Once expression levels and function are characterized, constructs will be selected for stable transfection. Freestyle NS-293 cells will be used for both transient expression and/or stable transfection. A BioRad Gene Pulsar will be used to transform the cells. Once transformed the cells will be plated in a single flask as a master bulk culture. The culture will rest for 72 hours followed by continuous dilution of this culture to allow subcloning into single cells. This will be diluted into 96 well plates containing a neomycin selection reagent.The supernatants from the 96 well cultures of first subcloning, will be tested for specific protein expression of GLP-2 or ghrelin and RJW2 using high throughput ELISAs for GLP-2, ghrelin, and RJW2. We will develop assays that will only detect the intact fusion protein using a sandwich ELISA.Although we will use the proteins produced by the semi-clonal method in most of our assays we will also produce monoclonal cell-lines. The downstream process will involve optimization of HIS tag affinity purification, and if necessary, additional polishing of the protein by ion exchange chromatography. The finished high purity protein will be buffer exchanged using a Hitrap desalting column, concentration calculated using UV 280nm spectrophotometry by Nanodrop, and characterized for product size and purity by SDS PAGE. Finished protein will be packaged and stored at 4oC and tested for function and stability. Each of the proteins will be evaluated by ELISA and for biological activity using the specific bioassays for GLP-2 and ghrelin activity.For Specific Objective 2, a specific bioassay for ghrelin activity will be developed using HEK 293 cells that have a stable expression of the GHS-R1a receptor (ghrelin receptor). As positive controls, native ghrelin or commercially-available ghrelin receptor agonists will be evaluated. Each of the purified recombinant Scienss ghrelin fusion proteins will be evaluated for in vitro biological activity to assure that the recombinant proteins bind to the ghrelin receptors and activate second messenger pathways (cAMP). These bioassays will utilize a positive control (dose response of commercially-available ghrelin receptor agonist) and a negative control (recombinant RJW2 protein without attached ghrelin).For Specific Objective 3, we will utilize an in vivo bioassay for gherlin in mice. The key changes that will be measured are feed intake and weight gain.A total of 24 mice will be utilized in this experiment. During two weeks of treatments, mice will be evaluated daily for feed intake and for weight gain. It is anticipated that ghrelin will increase appetite and feed intake and thereby increase weight gain.We plan to use methods that are very similar to the well-characterized methods for measuring GLP-2 activity in mice. We will utilize an in vivo bioassay for GLP-2 in mice. The key change that will be measured is weight of the intestine in control and mice treated with GLP-2. A total of 24 mice will be utilized in this experiment. This experiment is not designed to evaluate the optimal dose of the protein needed for the intestinotrophic effects of GLP-2 but is designed to evaluate whether the GLP-2 fusion protein has in vivo GLP-2 like activity.We will also determine half-life of the recombinant ghrelin proteins in sheep. We will determine in vivo biological activity of ghrelin proteins in sheep using GH secretion, feed intake, and weight gain. For this task, prepubertal lambs (~6 months old) will be utilized.We will also determine the half-life of recombinant GLP-2 proteins in sheep and determine the biological activity of GLP-2 in sheep. The design will be very similar to the design with ghrelin. Prepubertal lambs (~6 months old) will be utilized.