| Severe acute respiratory syndrome (SARS), also named infectious atypical pneumonias, spread worldwide as a transmissible febrile respiratory disease in 2002/2003. Currently, a SARS-associated coronavirus (SARS-CoV) was identified as the etiological pathogen of this disease. SARS-CoV is a newly emerging member of coronavirus family, whose virulence is higher than other coronavirus. Today, There are still many features of SARS-CoV remain unknown. These indicate that SARS epidemics may recur at any time in the future. Therefore, development of the susceptible, specific diagnosis methods and safe, effective vaccines is urgently needed for protection of at-risk populations. Currently, one candidate vaccine using inactivated SARS-CoV is in a phase I clinical trial in China, and the serological diagnostic kits for SARS has been commercialized. However, there has been a serious concern about their safety since the infectious mRNA in virus particles may pose serious risk, and some antigens of the virions may elicit antibodies that do not neutralize, but rather enhance virus infection, that is the hindrance for SARS-inactivated vaccine study. Moreover, it's remain a serious dangerous work to operate such a contagious virus even though in biosafety level 3 laboratory, since there have no vaccine and therapeutic medicine currently. So it is a hot point in study of recombinant protein-based diagnostics and genetic engineering subunit vaccine for SARS by biotechnology methods. Spike glycoprotein (S protein) of SARS-CoV is a most important class I transmembrane glycoprotein among membrance proteins, it is responsible for virus binding, fusion and entry, and is the major inducers of neutralizing antibodies. Besides, it plays critical roles in viral pathogenesis and virulence. SARS-CoV S protein contains a number of antigen epitopes, So there is a high level of antibodies specific to S protein in SARS convalescent phase sera, which emerging earlier and lasting longer. So detection of sera antibodies to S protein is also a diagnostic marker for SARS. Nucleoprotein (N) is also a major immunogenetic antigen for SARS; however, several tests have shown that N protein had the reactivity with other coronavirus positive sera. It is therefore an advantage of S protein as recombinant target antigen for SARS serum diagnosis compared with recombinant N protein. Moreover, S protein has been shown to be the major antigenic determinant that induces neutralizing antibodies and protective immunity to SARS-CoV, so it is also important in designing SARS genetic engineering vaccine. It is difficult to express full-length S gene in vitro as it contains 1255 amino acids, even though it can be expressed, the recombinant protein has low solubility; The second, the tests confirmed that the truncated S protein had a higher positive ratio than full-length S protein in detection of SARS positive sera, the reason is that the interference of many non-neutralizing epitopes on full-length S protein. These non-neutralizing epitopes may elicit enhancing antibodies and induce human excessively allergy reaction, which is a possible factor in pathogenesis of SARS. Considering these reasons, it is necessary to select S protein fragments as candidate antigen for diagnosis, which contains major antigen epitopes and can recognize S protein-specific antibody in sera. At the same time, the study of immunogenicity of S protein fragments also help to carrying out the subunit vaccine study of SARS. SARS-CoV S protein is a highly glycosylated protein, it is therefore being suitable expressed in eukaryotic system. In this study, we use insect cell-baculovirus system to express eight gene segments overlapping the full length S gene of SARS-CoV. Firstly, eight genes were sub-cloned into pFastBacHTa vector respectively and the recombinant pFastBacHTa-SX plasmids were identified by PCR and endonucleases digestion (X represents the name of S gene segments: A,B,C,D,E,F,G1,G3). The rpFastBacHTa-SX plasmids were transported into DH10BacTM competent cells, then the recombinant Bacmid DNA were extracted and identified by PCR using PUC/M13 primers. The recombinant bacmids were used to transfecte s.frugiperda (Sf9) cells to obtain the recombinant baculovirus. For protein expression analysis, Sf9 cells were harvested and lysised when cell CPE (cytopathogenic effect) were observed. Total cells lysate from each infection was submitted to SDS-PAGE analysis and the expression proteins were confirmed by Western blotting with rabbits positive single-factor sera immunized with S protein fragments expressed in E.coli. The results showed that the eight gene segments were specifically expressed in Sf9 cells with the expected sizes and the expression proteins had immunoreactivity in immunoblot assay. So we obtained 8 strains of recombinant baculovirus contain 8 gene segments of SARS-CoV S gene, which were named rBaculovirus-SX. Immunoblot assay was used to identify the antigenic of S proteins fragments expressed in Sf9 cells with SARS positive polyclonal antibodies. We selected 4 recombinant proteins that reacted with 2 portions SARS convalescent-phase sera and 1 portion SARS-positive yolk antibody. These 4 proteins are recombinant SF, SG3, SA and SB proteins. To be used as indirect ELISA(iELISA) coating antigen, Sf9 cells was inoculated with rBaculovirus-SF,SG3,SA,SB with 5-10 MOI respectively, the supernatant was obtained form infected cells by washing, ultrasonic lysis and centrifuge, which was used as coating antigen, then the iELISA method was established using rabbit single-factor sera. The SARS-positive sera were detected using iELISA method and the results showed that recombinant SF, SA, SB protein had nonspecific reaction with the sera, so they cannot be used as iELISA antigen directly. The recombinant SG3 protein-based iELISA was selected by its specificity and reactivity, then was compared with the commercial kits by detection SARS-positive sera. The results showed that the two methods have an identity on SARS-positive sera end-point and seropositivity detection. In order to use expressed SG3 protein as diagnostic antigen, we optimalized the expression factors of SG3 protein to improve its solubility and detected its thermostability. This study was the basic work to recombinant SG3 protein being used as diagnostic antigen in SARS serological diagnosis. To carry out the immunoflourescence assay (IFA), the Sf9 cells were infected with eightrecombinant viruses respectively and harvested at about 60h postinfection. The antigen plates were prepared using infected Sf9 cells, which were used to detect the rabbits single-factor sera, and the titers showed ranging from 1:400 to 1:1600 respectively, while the normal rabbit negative serum had no nonspecific reaction with these antigens. We tested the reactivity of 2 portions SARS-positive sera and 1 portion SARS-positive yolk antibody with eight antigen plates, the results showed that, except the SF,SG3,SA,SB proteins, the SC and SE proteins had immunoreactivity also. NO cross-reactivity was founded with serum against infectious bronchitis virus (IBV), mouse infectious hepatitis virus (MHV) and 5 portions normal human sera in our assays. These results indicated that the potential application of the IFA based on rBaculovirus-SF and rBaculovirus-SG3 infected Sf9 cell. One question is that the sera samples using in our study are too little to obtain statistical standard, so it is necessary to obtain enough SARS-positive sera samples to testify these methods in future study. In summary, our Spike protein fragments-based indirect ELISA and IFA methods could offer a safer procedure, user-friendly and cost-effective than the whole virus base-sera detection method, they can be used in SARS diagnosis as supplementary or distinguishing method. In addition, eight gene segments overlapping the full-length SARS-CoV S gene expressed in eukarytic system can be used in immunogenicity, antigenicity and function study of S protein, which can promote the study of subunit vaccine and therapy to SARS, and also the function study to SARS-CoV Spike glycoprotein.
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