Expression Of SARS-CoV Non-structural Protein And Its Application In Assaying Corresponding Antibodies From Patient's Serum

Severe acute respiratory syndrome (SARS) is a newly emerged disease attracted global attentation because of its highly contagious character in the increasingly globalization. The origination of SARS is Guangdong Province of China. The first outbreaks affected over 8,000 people and spread to more than 29 countries which causede more than 900 people died. It is now understood that a novel coronavirus, SARS-CoV, is the etiological agent for SARS. The SARS-CoV is a single-strand, positive-sense RNA virus, about 29Kbp in length, with the characteristic gene order [5'replicase (rep), spike (S), envelope (E), membrane (M), nu-cleocapsid (N) 3']. Besides the four structural gene, S,E,M,N, there are some nonstructural gene such as 3a, 3b and 7a are found. Which locate bettween S E and M N, the function of these nonstructual genes is still unknow. In this study, nonstructural gene 3a, 3b and the truncate, 3a_157-274aa, 3b_81-154aa and the structural gene nucleocapsid (N)were subcloned into pGEX6P-l and pET28a respcetively and then were transformed into BL21(DE3) E.coli, after induced to to express by IPG, These proteins were purified by SDS-PAGE or affinity chromatogrphy, then identified by western-blot. We detect antibody in serum from 10 SARS patients with 3a-GST, 3b_81-154aa-GST, N-HIS and GST protein as diagnostic antigen respectively by indirect ELISA. The ELISA result suggested that there existed the antibody of N and may there are the antibody of 3a and 3b in patient's serum. To avoid the cross-reaction between patient's serum and GST protein, we transfer 3a_157-274aa, 3b _81-154aa and 7a_17-101aa genes to the expression vector pET32a.Recombinant were induced to express by IPTG The recombinant fusion protein was hydrolyzed by thrombin to remove the trxA away. Then serum from 21 clinically confirmed SARS case and 21 normal subjects were tested against N, 3a_157-274aa, 3b_81-154aa and 7a_17-101aa proteins as antigen respectively. 20 case have strong response to N protein, positive rate is 95.2%, to 3a_157-274aa ,16 case have strong response ,positive rate is 76.2%, to 7a_17-101aa. 12...