western blot protocol pdf Archives - Gen9 Genetics

To identify an important antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the The full-length and six truncated fragments of the nucleocapsid gene were expressed and purified as recombinant glutathione S-transferase-tagged proteins. The reactivities of the recombinant proteins were examined against a panel of antibodies containing 33 positive sera for SARS coronavirus and 66 negative sera and against antibodies against other animal coronaviruses.

A 195 amino acid truncated fragment of the C-terminus of the nucleocapsid protein (N195) was identified as having a strong ability to detect antibodies against the SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies to the SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blot assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot and IFA assay for the detection of SARS coronavirus positive sera were the same. Therefore, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.

Coronaviruses are the largest enveloped positive strand RNA viruses, with genome sizes ranging from 27 to 30 kb (8,13). The genomic organization of the SARS coronavirus is typical of coronaviruses, with a characteristic genetic order (rep-licase, peak [S], envelope [E], membrane [M], and nucleocap-sid [N]). Structural proteins (S, E, M, and N) function during host cell entry and virion morphogenesis and release.

During the assembly of the coronavirus virion, N binds to a defined packaging signal in the viral RNA, leading to the formation of a helical nucleocapsid. N also has a novel nuclear function, which could play a role in pathogenesis. Based on previous findings, N was identified as the target gene for the development of a diagnostic PCR (4, 6, 18). The N protein of coronaviruses (such as infectious bronchitis virus [IBV]) is highly conserved in each group, it is immunogenic,