Matching Items (1)
Description
The coronavirus envelope (E) protein is a structural component of all coronavirus. Obtaining purified E protein in an efficient, clean, and reliable manner is needed for ongoing studies. Toward this goal the E protein of Mouse Hepatitis Virus (MHV) coronavirus was tagged with with either 6 or 10 histidine (his) residues which can be used for affinity chromatography purification. The his-tags were introduced by PCR into a cDNA of the MHV A59 virus strain at the carboxy end of the E gene. A reverse genetics approach was then used to assemble three full-length cDNAs of the viral genome, two modified with the hig-tags and a control wild-type (WT) without a tag . Full-length genomic RNAs were transcribed and electroporated into baby hamster kidney cells that express the MHV receptor (BHKR) and L2 rat lung cells. Virus was recovered after 72 h only from the 6X his-tagged genome and the WT control. Western blotting using antibodies against the E protein or the nucleocapsid (N) protein was performed after cells were infected with the recovered WT and 6X-tagged recombinant viruses. The E protein was not detected with the E antibody, but was detected with a histidine probe was used to detect the histidine residues. This indicates that the tagged protein is expressed and that the tag is present.
ContributorsHesser, Kathryn Sarah (Author) / Hogue, Brenda G. (Thesis director) / Hogue, Ian B. (Committee member) / Lim, Efram (Committee member) / Dean, W.P. Carey School of Business (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05