gms | German Medical Science

VIRCELL, SL is a spanish company interested in the development of diagnostic kits for emerging diseases. Lately, 4 recombinant proteins and a polyclonal antibody specific for the novel coronavirus associated with the severe acute respiratory syndrome (SARS), have been developed. The recombinants were designed as parts of two different antigens from SARS-CoV, identified thanks to their similarity with described proteins related to other well-characterised coronavirus. Firstly, one of the recombinants is a polypeptide for the spike (S) glycoprotein, a major target of the cellular immune response to coronaviruses. It plays an important role in the initial stages of infection due to it forms the characteristic corona of large, distinctive spikes in the viral envelopes. This viral genomic sequence is a fragment of 620 bp which express a 27 kDa-protein with potential epitopes detected (i) by using a theoretical software named EMBOSS and (ii) by comparation with other spike proteins from antigenically characterised coronavirus. Secondly, the other 3 cloned DNA fragments belong to the nucleocapsid protein N, described by Krokhin et al. [Ref. 1]as the major immunogen in the SARS-CoV, which could be useful for early diagnostics. These fragments named as N1, N2, N3, express respectively an 18 kDa-, 15 kDa- and 20 kDa-protein and involve the nucleocapside protein in its entirety. The 4 fragments were amplified by RT-PCR from the viral RNA and cloned into the vectors pRSET and pET-15b in order to perform their expression in Escherichia coli, specifically the strain Rosetta BL21 (DE3) pLysS, which enhances the expression of eukaryotic protein that contains codons rarely used in E. coli. The 4 recombinants are expressed in more than 30% by adding the inductor, IPTG, and contain a His-tag in their amino-terminal, which allows purification by Ni affinity in more than 98%. These four recombinants have allowed us to obtain a specific polyclonal Ab. All of them have been analysed against positive sera, obtaining very good results with a mixture of them. The Ab has been checked against an extract of cells infected by SARS-CoV, obtaining a clear positive in immunofluorescence. At this moment, the same recombinants are being developed in baculovirus expression system by transfection in Sf9 cells in order to use them in an IFI diagnostic kit and monoclonal Ab have been developed.