Cloning And Expression Of SARS-COV Spike Glycoprotein Gene Segments

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,Today。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.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.The main protein of SARS coronavirus are RNA polymerase,S protein,E protein,N protein,in which the S protein is maining in change of the infection.The difference of S protein will not only cause the change of the host,but will also lead to the cold, peritonitis,gastroenteritis and many diseases of the host.Meanwhile,S protein is also the main part which influences the level of the infection.Referring to the sequence of S protein published by GeneBank,We designed a set of primer.The PCR amplification product of A protein was analyzed by agarose gel electrophoresis and demonstrated to be a specific strain about 576bp.The recycle was cloned into the and digested by BamHⅠandXbaⅠThen We recycled the target fragments,ligated them repectively to the expression vector with T4 ligase.which is digested by the same two enzymes,and We also further sequenced it and analyzed its autoploidy.After the cloning the A genes into the expression vector , further digesting by BamHⅠand XbaⅠand identification,We finally got the positive expression vector.The host BL21(DE3) was induced by IPTG with the concentration of 2 mmol/L.The amount of expression counts percent of the whole proteins By Western blotting.the proteins expressed can be detected by SARS positive serum.which will contribute greatly to the establishment of SARS antibody detecting method.Using the A protein expressed as antigen。We amplified the B genes of S protein by PCR,cloned the B genes of S protein and then established the eukaryotic expression vector,which is the basis of the further study and produce of nucleic acid bacterin.