Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus called SARS-CoV (7, 8, 10). The virus causes atypical pneumonia with diffuse alveolar damage with an overall mortality of 10% that ranges from 0% in children and 50% in persons over 65,seriously threatening public health worldwide.
The SARS-CoV is an enveloped virus of a positive single stranded RNA genome~29.7 kb in length and has 11 open reading frames. The genome organization is similar to that of other coronavirus,but phylogenic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronavirus (7, 10). The structural protein genes of SARS-CoV are contained in four major open reading frames,and include the surface spike(S) glycoprotein,the small membrane protein(M),the envelope(E) glycoprotein,the nucleocapsid(N) protein,and a set of accessory proteins whose number and sequence vary among different coro-naviruses (11).
The S protein of SARS-CoV is a large type Ⅰ membrane glycoprotein projection from the viral envelope which is responsible for both binding to receptors on host cells and for membrane fusion. Angiotensin converting enzyme 2(ACE2) was found to be an efficient receptor for the S glycoprotein of SARS-CoV (9, 4, 12). The S protein also contains important virus neutralizing epitopes that elicit neutralizing antibody in the host species (3, 2). To determine the critical sequence of the S protein that interacts with the ACE2 receptor,the interactions between ACE2 and different truncated S proteins were investigated. Our study sheds some light on the interaction mechanism and provides useful insight into the development of a protein vaccine for SARS-CoV.
The fragments of 1 035bp (S1-1),879bp (S1-2),657bp (S1-3),222bp (S1-4),705bp (S1-5),128bp (S1-6) and 327bp (S1-7) were amplified by PCR using plasmid pGEM-S as template and were then cloned into prokaryotic expression vector pETHis. The corr-ected clones were confirmed by restriction analysis and sequencing. The results of agarose electrophoresis are shown in Fig. 2 and Fig. 3.
Figure 2. Analysis of recombinant plasmid pETHis S1-1~S1-6 by BamHⅠ﹠EcoRⅠ digestion M,1kb DNA ladder; 1,pETHis S1-1; 2,pETHis S1-2; 3,pETHis S1-3; 4,pETHis S1-4; 5,pETHis S1-5; 6,pETHis S1-6; 7,pETHis S1-5.
S1-1~S1-7 proteins were successfully expressed in E.coli BL21(DE3) at a high level and formed insoluble inclusion bodies. As Fig. 4 shows,the proteins were expressed and purified to a high purity.
To investigate whether the induced proteins were the expected proteins,Western Blot analysis of purified proteins were processed. As Fig. 5 showed,all purified S1-1~S1-7 proteins could react with the SARS pat ients sera specifically and the molecular weights were consistent with expectations,indicating the approp-riate proteins were produced.
The recombinant ACE2 protein was successfully expressed in E.coli DH5. Fig. 6 showed that ACE2 was induced and purified. The purified ACE2 protein could react with anti-ACE2 serum and the expected 92kDa band was observed in Western Blot (Fig. 7).
Figure 6. Expression and purification of the recombinant protein ACE2. M,protein marker; 1,Cell lysate of DH5α carrying plasmid pProEXHTb after induced 6h; 2,cell lysate of DH5α carrying plasmid pProEXHTb-vero-ACE2 after induced 6h; 3,Purified recombinant protein ACE2.
Purified recombinant proteins S1-1~S1-7 were fixed in the 96-well plate by coating,and then purified ACE2 protein was added to allow their interaction. A well coated with purified ACE2 protein was used as positive control. Another coated with purified S1-1 protein without adding ACE2 protein was used as negative control. Then Anti-vero-ACE2 rabbit serum was added as first antibody and HRP labeled goat-anti-rabbit IgG was added as second antibody. ELISA showed that the results of S1-1,S1-2,S1-3,S1-5,S1-7 wells were positive,while the results of S1-4,S1-6 were negative (Table 1.). Our data showed that the aa 388~396 of S1 protein was essential to the interaction between S protein and ACE2.
Table 1. Interaction between truncated S protein and ACE2
To further confirm this result,an antibody induced by S1-7 which only contains aa 388~396 of S protein was used for disrupting the interaction between S and ACE2. The results show that all the interactions we re nearly completely blocked by this antibody (Table 2.). Whereas the antibody induced by S1-4 which contained deletions aa 388~396 did not show ability to block this interaction.
Table 2. Interaction blocking by antibody against S1-7