Cloning, sequencing and expressing in E.coli for a SARS-Cov N gene founed in Shanghai

  Objective   To analysis the mutation of SARS-Cov Ngene and its application in diagnostic kits by cloning, sequencing and expressing of Ngene from a Shanghai SARS-Cov strain.

  Methods   The SARS-Cov Ngene was amplified by RT-PCR fr om serum in a Shang hai SARS patientand was cloned into plasmid pGEX-2T.The cloned Ngene was sequenced and compared with 89 SARS strains in GenBank.The cloned gene was expressed in E.coli.The products was purified by affinty chromatog raphy of Glutathion-e Sepharose 4B and estimated by SDS-PAGE.

  Results   SARS-Cov Ngene 1269 bp was amplified and cloned.Sequencing analisis founded 1~4 bp mutation and 1~3 putative amino acid changed among amplified SARS-Cov Ngene and 89 SARS strains in GenBank.In E.coli, the Nprotein was expressed in 74000 Da fusion protein.The purified products was purity over 90%.

  Conclusion   99.92~99.68% nucleotides, 99.8~99.3% of putative amino acid were homologous among amplyfied Ngene and 89 SARS-Cov strains in GenBank.The basic data and protein products with high purity for diagnostic kitwas obtainded.