Researcher(s)
- Isabella Rogozinski, Pre-Veterinary Medicine and Animal Biosciences, University of Delaware
Faculty Mentor(s)
- Mark Parcells, Animal and Food Sciences, University of Delaware
Abstract
For the study of many emerging human and insect viruses, there is currently a lack of antibody reagents. A rapid, humane, and effective method for generating antibodies to study and treat viral infections is the production of antigen-specific immunoglobulin Y (IgY) from the yolks of chickens immunized with purified viral proteins. We have cloned the nucleocapsid (NC) and capsid genes from Heartland virus (HRTV), an emerging tick-borne pathogen first identified in Missouri in 2009 (1). HRTV causes flu-like symptoms that can persist for days or even months in severe cases. With rising global temperatures, the risk of HRTV transmission is expected to increase. The virus has three negative-sense RNA segments: small (S), medium (M), and large (L).
The NC protein is encoded by the S segment and was cloned into a bacterial expression vector, pMal-C2-Xa-eGFP-TEV. This vector encodes a fusion protein consisting of maltose-binding protein (MBP), enhanced green fluorescent protein (eGFP), and the NC protein, separated by Factor Xa and TEV protease cleavage sites. Gene-specific primers were designed to add a unique Nhe I site at the 5′-end and a 6×His tag with a Hind III site at the 3′-end. PCR-amplified products were TOPO-cloned into pCR4.0, and E. coli was transformed with the resulting plasmids. Positive clones were screened by restriction digestion and sequencing.
Confirmed inserts were subcloned into the expression vector using Nhe I and Hind III digestion. These constructs were transformed into E. coli, screened, and sequence-verified. The final vectors were introduced into Rosetta (DE3, LysS) E. coli for high-yield protein expression. Cultures were grown overnight and induced with 1 mM IPTG. Protein expression was monitored by collecting samples at 0, 1, 2, 3, and 4 hours post-induction to assess eGFP fluorescence.