Study Of Specific Antibody/Antigen Genes And N Gene Of SARS-CoV

Severe acute respiratory syndrome (SARS) with a significant morbidity and mortality, caused by a previously unidentied coronavirus termed SARS-CoV, was found first in the Foshan City, Guangdong Province, South China, on 16 November 2003, then promptly swept across the globe, with reported cases in more than 30 countries or regions. Without a "good standard" and reliable, early detect or diagnosis tests for diagnosing patients and monitoring its spread, as well as effective vaccines and antiviral compounds for preventing or treating this disease, it had resulted in over 800 deaths from more than 8000 probable cases worldwide. Although SARS was eradicated by isolation towards the end of 2003. sporadic cases have been reported in Singapore, Taiwan and mainland China, indicating that SARS maybe emerge again in the future. In this review, SARS is discussed as a disease, as well as its diagnosis, management and pharmacotherapy.Recently, the studies on detection and diagnosis of SARS infection mainly include as follows: Immunoassay based on Antigen-antibody reaction, viral genome detection, virus isolation and gene chip technology. For the immunoassay, it always includes two sides: antigen-specific detection and antibody-specific detection. However, ELISA (Enzyme-linked immunosorbent assay) may be the most effectiveimmunoassay for defection or diagnosis of SARS infection. To develop early detect or diagnosis reagents, our works were mainly involved in two sides as follows:One is to pan the specific scFv-antibody against SARS CoV, which facilitates detection of SARS CoV by ELISA. The human single fold single-chain antibody fragments (scFv) libraries I+J (Tomlinson I+J) was used to identify novel scFvs, which can specifically bind to SARS-CoV. Interestingly, two scFvs (B5 and B9) exhibited higher binding specificity to SARS-CoV with the OD450 value 0.608 and 0.545, respectively, and their coding sequences shared the identical sequence composed of VH gene (351 bp) and VL gene (327 bp), so the two scFvs were uniformly named as SA59B and chosen for further analysis. SA59B scFv was expressed in soluble form in E.coli HB2151 and purified by immobilized metal affinity chromatography (IMAC). The soluble 30 kDa SA59B scFv-antibody was verified in SDS-PAGE and Western-blot. The purified SA59B scFv-antibody was labeled with HRP by the glutaraldehyde method, and the concentration of HRP and SA59B scFv-antibody in the SA59B-HRP solution reached 2.4 mg/ml and 2.28 mg/ml, respectively. Then, the binding ability of SA59B-HRP to SARS-CoV was evaluated by ELISA with S/N of 11.6, indicating higher binding specificity between them. Finally, both the SA59B sequence specificity and its application for diagnosis, prophylaxis or therapy of SARS were discussed.The other is to construct gene engineering subunit-antigen of SARS-CoV S protein for detection of antibodies produced in response to infection with SARS-CoV. The antigenic analysis of SARS-CoV S protein by Vector NTI 8.0 reveals that it is not only involved in lipid-membrane fusion and viral nucleocapsid entry, but also is a main antigen of SARS-CoV, and that its antigenic regions mainly located in the COOH-terminal S2 subunit. The gene fragment encoding the 321 residues in the COOH-terminal of Spike proteins (termed S2-321) was amplified according to the sequence of SARS coronavirus TOR2 (AY274119) by nested RT-PCR, and cloned into prokaryotic vector pET28. The vector with S2-321 was used to transform E. coli BL21(DE3), the S2-321 polypeptides were then expressed in E. coli BL21(DE3) and purified by IMAC. The results of SDS/PAGE and Western-blotting suggested that theS2-321 polypeptide has about 33kD molecular weight, which can be detected by anti-6>7, GGWFCP7 ) in the NH2-terminal of the 15-mer peptides were just located in the [2Fe-2S] redox centre (including four conserved cysteines) of UCRI, indicating higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Finally, the possible mechanism of apoptosis-inducing of the N protein of SARS-CoV was predicted in two levels based on the possible interaction of the N protein with UCRI: 1) The N protein of SARS-CoV may be inhibit electron transfer in the mitochondrial respiratory chain at the levels of complex III(Ubiquinol-cytochrome C reductase), which lead to overstock of cytochrome C in the mitochondrial respiratory chain, the release of cytochrome C then trigger apoptosis. 2) The N protein of SARS-CoV may be inhibit ATP synthesis in mitochondrial energy metabolize level, which causes onset of the mitochondrial permeability transition (MPT) and the dissipation of the mitochondrial transmembrane potential, as well as the translocation of cytochrome C and apoptosis-inducing factor (AIF) from mitochondria to an extra-mitochondria! localization, thereby causing apoptosis.Currently, efforts are underway to further investigate the phenomena and mechanism of apoptosis induced by the N protein, which may be useful for investigating the relation of release of cytochrome C or AIF and electron transfer or energy metabolize in apoptotic signal transduction pathway mediated by mitochondria.