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目的克隆SARS冠状病毒(SARS-CoV)GD322株M基因,并进行序列分析。方法根据GenBank中公布的SARS冠状病毒Tor2株M基因序列,设计一对引物,用RT-PCR法从SARS-CoVGD322株基因组中扩增M基因片段。克隆至pET-32(a)载体,转化大肠杆菌BL-21后测序,利用DNAstar和ClustalX分析所测序列翻译的氨基酸与81株SARS-CoVM基因翻译的氨基酸序列的差异。结果该M基因与Tor2株M基因核苷酸同源性为99.86%;与已收集的81株SARS-CoVM基因所译氨基酸相比,和41株(占50.62%)M蛋白完全同源,37株(占45.68%)仅有一个氨基酸改变,同源性为99.55%,仅与3株(占3.70%)有2个氨基酸差异,同源性为99.10%。结论已获得具有代表性的SARS-CoVM基因重组质粒。
Objective To clone the M gene of GD322 strain of SARS coronavirus (SARS-CoV) and analyze its sequence. Methods According to the sequence of M gene of SARS coronavirus Tor2 strain published in GenBank, a pair of primers was designed and the M gene fragment was amplified from the genome of SARS-CoVGD322 by RT-PCR. Cloned into pET-32 (a) vector and transformed into E.coli BL-21. DNAstar and ClustalX were used to analyze the differences between the translated amino acids and the amino acid sequences of 81 SARS-CoVM genes. Results The M gene was 99.86% homologous to the M gene of Tor2 strain. Compared with 81 strains of SARS-CoVM gene, 41 M strains (50.62%) were completely homologous to the M protein, 37 Strains (45.68%) had only one amino acid change, with 99.55% homology and only two amino acid differences with three strains (3.70%). The homology was 99.10%. Conclusion A representative recombinant plasmid of SARS-CoVM gene has been obtained.