2005•Jilin Nongye Daxue xuebaoRequires access
Cloning, sequence analysis and expression in E.coli of nucleoprotein gene of SARS coronavirus
Yang SongTao, Xianzhu Xia, Jun Qiao, Yuwei Gao, Shuang Chang, Geng Huang, Zheng Mingguang
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Abstract
Abstrcat Nucleoprotein(N) gene of SARS coronavirus (SARS-CoV) was successfully cloned and sequenced. The N protein gene was amplified by RT-PCR with a pair of specific primers, then cloned into pGEM-T and sequenced. The results of sequencing showed that the full length of N gene was 1 269 bp and encoded 422 amino acids. The homology of nucleic acids and amino acids were all 100%, respectively, compared with the standard strains Tor2, TW1 and Urbani. Then nucleoprotein gene was subcloned into the prokaryotic expressing vector pET28a. Positive recombinant was transformed into E. coli strain BL21(DE3) for expression under induction of IPTG. The results of SDS-PAGE revealed that the molecular weight of expression product was 53 kD, which could be specifically recognized by polyclonic antibody against SARS virus through Western blotting analysis. The expression of recombinant N protein of SARS could amount to 43% in the total protein of the induced bacteria by gel scanning analysis.