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. 2006 Jul 10;580(16):3799-803.
doi: 10.1016/j.febslet.2006.06.002. Epub 2006 Jun 12.

Amino acids 15-28 in the ectodomain of SARS coronavirus 3a protein induces neutralizing antibodies

Sara Akerström  1 , Yee-Joo Tan, Ali Mirazimi
Affiliations

Amino acids 15-28 in the ectodomain of SARS coronavirus 3a protein induces neutralizing antibodies

Sara Akerström et al. FEBS Lett. .

Abstract

A synthetic peptide corresponding to amino acids (aa) 15-28 of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein was used to raise polyclonal antibodies in rabbits. This anti-3a N-terminal antibody could detect 3a protein in infected cells, as did an anti-3a C-terminal antibody previously described. The latter targeted the C-terminal cytoplasmic domain of 3a (aa 134-274). The anti-3a N-terminal antibody could detect intracellular 3a as well as 3a expressed on the cell surface. Interestingly, only the anti-3a N-terminal antibody can inhibit SARS-CoV propagation in Vero E6 culture although the binding affinity of the anti-3a N-terminal antibody was lower than the anti-3a C-terminal antibody.

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Figures

Figure 1
Figure 1
Detection of SARS‐CoV 3a protein expressed in Vero E6 cells with rabbit anti‐3a polyclonal antibodies. (A) Rabbit #2 and (B) Rabbit #3: Western blot analysis was performed using sera obtained from two rabbits that were immunized with a synthetic peptide which corresponds to amino acids 15–28 of the 3a protein (i.e. N‐terminal). All sera were diluted 1:2000 and the lysates were obtained from Vero E6 cells transiently transfected with a DNA construct for expressing full‐length 3a (+) or untransfected cells (−). (C) Vero E6 cells were infected with an MOI of 1 and then prepared for Western blot analysis. Pre‐immune serum from the rabbit (Rabbit #2) that was immunized with the 3a N‐terminal peptide showed no reactivity (lane 1), while the 5th bleed from the same rabbit after immunization detected the 3a protein in infected cells (lane 2). Similarly, pre‐immune serum from the rabbit (Rabbit #1) that was immunized with the 3a C‐terminal bacterially expressed protein showed no reactivity (lane 3), while the 6th bleed from the same rabbit after immunization detected the 3a protein in infected cells (lane 4). (D) Cellular localization of 3a in transfected Vero E6 cells as determined by indirect immunofluorescence. Serum from the 5th bleed of the rabbit (Rabbit #2) that was immunized with the 3a N‐terminal peptide was used at a dilution of 1:200 to detect for the 3a protein expressed on the cell surface (left panel, no permeabilization) and intracellularly (right panel, permeabilization with 0.2% Triton‐X 100).
Figure 2
Figure 2
Binding affinity of rabbit polyclonal antibodies to 3a protein expressed in 293T cells as determined by ELISA. Rabbit sera were diluted 1:2000 (A) or 1:8000 (B) and added to wells coated with total cell lysates from untransfected cells or cells transiently transfected with a DNA construct for expressing the full‐length 3a protein. The OD difference (in arbitrary units) represented the specific binding of antibody to the 3a protein. Rabbit #1 was immunized with bacterially expressed GST‐3a (134–274 aa) (i.e. C‐terminal) and the pre‐immune serum and the serum from the 6th bleed were tested. Rabbit #2 was immunized with a synthetic peptide corresponding to amino acids 15–28 of 3a (i.e. N‐terminal). For Rabbit #2, the pre‐immune serum and the sera from the 1st to 5th bleeds were tested. All experiments were performed in duplicates and the average values with standard deviations are plotted.
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