Expression Of N-Protein From SARS Associated Coronavirus (SARS-CoV) And Establishment Of Serodiagnostic Method

Objective: This study aimed to express and purify recombinant nucleocapsid (N)- Protein from SARS associated coronavirus (SARS-Cov) and develop an indirect enzyme-linked immunosorbent assay (ELISA) based on N fusion proteins for serodiagnosis of SARS-CoV infection.Methods: The N gene were cloned into pQE-30/N and expressed in Ml5 strains of Escherichia coli. The immunogenicity of N protein was analyzed by Western blotting using serologically confirmed sera from patients with SARS. Recombinant N protein was used as coating antigen for establishment of an indirect enzyme-linked immunosorbent assay. An ELISA based on N protein for IgG detection was tested with serum form 745 healthy blood donors and with serum from 404 patients with SARS at different time points of acute and convalescent-phases.Results: pQE-30/N encoding nucleocapsid fusion protein gene was successfully constructed. Expression of 6-His-tagged recombinant N protein were observed in E. coli M15 treated with IPTG. The N protein was successfully expressed and purified. The SDS-PAGE electrophoresis of the purified fusion proteins appear that the purity of N fusion proteins account for more than 90%. Western blot results showed N fusion proteins reacted with the sera of 12 SARS patients. A prominent immunoreactive protein bands about 47 kDa was seen in the Western blotting. An ELISA was developed for the detection of specific antibodies against N protein. Box titration was carried out with different dilutions of N protein coating antigen and peroxidase-conjugated goat anti-huamn IgG. The results identified the N proteins at concentrations of 0.1ug per well by incubation overnight (16h) at4C temperature, and the conjugated at concentration of 1:4000 as the ideal amount for IgG detection. To establish the cut-off value for the test, serum samples from 548 healthy blood donors who donated blood 2 years ago were test in ELISA. An absorbance value of 0.263 was selected as the cut-off value (the mean value for the healthy controls plus 5 SD). With seroconversion confirmed SARS patients as the gold standard, this assay had a sensitivity of 100% for patients on day 15 after the onset of symptoms, in contrast to ELISA with lysates of mutated strain of SARS-CoV as coating antigens. The results suggested the positive rates for N protein specific antibodies IgG increased with time in the course of disease. The test employs recombinant N protein rather than the cell culture extract of virus for serodiagnosis of SARS, it dose not require cultivation of SARS-CoV, and more biosafety than the virus lysates.Conclusion: The ELISA-based purification recombinant N protein from SARS-CoV appears to be a sensitive and specific test for serodiagnosis of SARS-CoV infection. The N protein would be used as antigens in the development of serological assay for detection of SARS-CoV infection.