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Title
Construction of a Recombinant Coronavirus with Histidine Tagged Envelope (E) Protein
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.
Date Created
2019-05
Contributors
- Hesser, 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)
Topical Subject
Resource Type
Extent
40 pages
Language
Copyright Statement
In Copyright
Primary Member of
Series
Academic Year 2018-2019
Handle
https://hdl.handle.net/2286/R.I.52896
Level of coding
minimal
Cataloging Standards
System Created
- 2019-04-20 12:04:37
System Modified
- 2021-08-11 04:09:57
- 2 years 8 months ago
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