Browsing by Subject "Coronaviren"

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Charakterisierung der akzessorischen Proteine vom felinen Coronavirus (FCoV) mit monoklonalen Antikörpern
(2014) Lemmermeyer, Tanja; Pfitzner, Artur J. P.
Little is known about the expression and function of the FCoV accessory proteins 3a, 3b, 3c, 7a and 7b. These proteins of FIPV strain 79-1146 were investigated in the present study. The obtained results are outlined: 1. The accessory FCoV proteins 3a, 3c and 7b were expressed in E. coli with a C-terminal his-tag. Furthermore the proteins 3a, 3b, 3c, the C-terminal half part of 3c, 7a, 7b as well as 7b without the amino acids 1 to 17 (7bdeltaSS) were expressed as fusion proteins with an N-terminal GST-tag and a C-terminal his-tag. The purification of all fusion proteins was performed by Ni2+ ion affinity chromatography under denaturing conditions. 2. To generate monoclonal antibodies the purified fusion proteins 3c and 7b were used for immunization of mice. ELISA screenings were established which enabled the identification of hybridoma cells that produce mabs against 3c and 7b. 3. The characterization of the anti-3c mabs led to the identification of regions in the C-terminus of the protein. The 3c protein could not be detected in an eukaryotic inducible expression system (Tet-on cell line BHKFIPV-3c) and also not in FCoV-infected cells. The anti-7b mabs bound within the region of amino acids 58 to 75 and reacted with a recombinant 7b fusion protein of a serotype I FCoV. 4. The expression of the 7b protein in infected cells was confirmed by western blot. An N-glycosylation site is located within the binding region. After incubation with tunicamycin the signal obtained with the anti-7b mabs was considerably stronger. Again after tunicamycin treatment the 7b protein was detected in the cytoplasm of infected cells by indirect immunofluorescence. The 7b protein colocalized partially with the ER. 5. The recombinant 7b protein was detected with all of the anti-FIPV 79-1146 sera and the ascites, but not with the anti-FECV 79-1683 sera. In contrast the recombinant fusion proteins 3a, 3b, 3c and 7a were not detected with the analyzed anti-FCoV sera.