| Severe acute respiratory syndrome （SARS） is a respiratory illness that has recently been reported in Asia, North America, and Europe. According to the World Health Organization, approximat ely 8,098 probable cases and 774 deaths from SARS have been reported worldwide as of September 26, 2003. Scientists from multiple laboratories throughout the world have detected a previously unrecognized coronavirus （called SARS CoV） in patients with SARS. This novel coronavirus is believed to be the cause of SARS. Laboratory assays for SARS-CoV were based on either the detection of the virus or virus products, or detection of an antibody response to viral infection. Current diagnostic methods for SARS-CoV were divided into three main aspects, molecular tests, virus isolation and antibody detection. SARS-CoV-specific RNA could be detected in various clinical specimens such as blood, stool, respiratory secretions or body tissues by RT-PCR. The presence of the infectious virus could also be detected by inoculating suitable cell cultures （e.g., Vero cells） with patient specimens and propagating the virus in vitro and identified as SARS-CoV using further tests. Cell culture is currently （with the exception of animal trials） the only means to show the existence of a live virus. Various methods provide a means for the detection of antibodies produced in response to infection with SARS-CoV. Enzyme-linked immunosorbent assay （ELISA） was a test detecting a mixture of IgM and IgG antibodies in the serum of SARS patients. Most of plates were coating with lysates of Vero cells infecting with SARS virus. Immunofluorescence assay （IFA） based on the microscope slide fixed with SARS-CoV-infected cells and immunofluorescent-labelled secondary antibodies. Neutralization test （NT） assessed and quantified the ability of patient sera to neutralize the infectivity of SARS-CoV on cell culture, by means of titration. Despite the rapid progress in the diagnostic methods of SARS infection, all tests currently available had limitations. The most remarkable one is the biosafety. The present investigation was undertaken to develop a serodiagnositic means which based on recombinant nucleocapsid protein of SARS coronavirus. Our findings suggest that the use of ELISA system and colloidal gold strips may present an effective approach to detect antibody against SARS-CoV without a BSL-3 condition. Part Ⅰ Over－expression and Antigenicity of Recombinant Nucleocapsid protein of SARS Coronavirus Objectives To obtain the Nucleocapsid protein （N protein） of SARS Coronavirus （SARS-CoV） conveniently and abundantly for detection the corresponding antibody in the serum and preparation of diagnostic kits. Methods Gene encoding N protein were separated into two parts according to the prediction of antigenicty. The recombinant plasmids were obtained by nucleotide synthesis and cloned into expression vector pET32a（+） respectively. Proteins then were induced to expression by IPTG and purified through Ni－NTA. Nealand rabbits were immunized with recombinant protein to investigate the antigenicity of recombinant protein. Results Analysis of antigenicity prediction revealed that N protein contained three strong determinants, which corresponded to 219~288, 390~399 and 157~167. The first part of N protein designed from 1 to 549 base pairs, that is 1~183aa, named N-1, and the second from 496 to 1269 base pairs, that is 166~422aa, named N-2. Target proteins, recombinant N-1 and N-2 were successfully expressed with relative molecular weight of 39,000 and 48,000 respectively. Both were purified via Ni-NTA with 85% purity. After immunized with recombinant N-1 the Zealand rabbit produced high titer polyclonal antibody. Conclusion The recombinant N protein of SARS-CoV possessed strong antigenicity, and could be very helpful to diagnose the disease and investigate the molecular characterization of virus further. |
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