Expression Of Neucleocapsid Protein From A SARS-CoV In E.Coli BL21 And Primary Application Of The Expressed Products

An outbreak of atypical pneumonia, designated severe acute respiratorysyndrome(SARS) by the World Health Organization(WHO) and first identified inGuangdong Province, China, has spread to several countries.The mortality of thisdisease is higher and therefore few hundreds of people have died from the disease.The cause of SARS has been identified as a novel coronavirus－ SARS-CoV.During a month, the genome sequencings for the coronaviruses from differentSARS patients have finished, which have been deposited in the GenBankalready(http://www.ncbi.nlm.nih.gov/). SARS-CoV is enveloped,positivesense,ssRNA virus. The genome ofSARS-CoV with 29727 nucleotides in length,has 11 open reading frames.Openreading frames(ORF) analysis through sequence similarity to the knowncoronaviruses indicated that several proteins coded by SARS genome might playimportant functions associated with SARS infection, including replicases 1a and1b,the spike(S) protein, the matix(M) protein, the nucleocapsid(N) protein and thesmall envelope(E) protein. To identify the functions of the SARS N protein, weare trying to over-express the important proteins of SARS and establish indirectELISA to assess serum antibody responses of patients with severe acuterepiratory syndrome to nucleocapsid antigen of SARS-associated coronavirus. In this research, genes encoding nucleocapsid(N) protein of SARScoronavirus were obtained by RT-PCR and were into expression vectorpET32a+.The recombinant plasmid were over-expressed in E.coli as inclusionbody with molecular weight 66KD which can specifically react with sera ofSARS patients by Western-blot and ELISA. Using the purified fusion protein, weimmunized normal rabbits for obtaining multiclonal antibodies and investigatedthe development of specific antibody against the novel coronavirus in patiens IIwith SARS by establishing indirect ELISA. The result of animal experiment andELISA showed that the fusion protein could stimulate animals to produceantibody and was specificity and sensitivity respectively. Then, we continued todevelop a rapid and efficient method for preparing monoclonal antibodies(McAb)against SARS-associated coronavirus N protein. This has laid a necessaryfoundation for further research on improving the detection rate of SARS-CoV.Thus,recombinant proteins constructed in this study may be considered aspotential candidates for the development of an SARS-CoV subunit caccine aswell as for the development of highly sensitive and specific diagnostic tests.