SARS冠状病毒M基因的克隆及序列分析

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。

Cloning and Sequences Analysis of SARS-CoV M Gene

Objective To clone and analyze the sequence of the SARS-CoV GD322 M gene. Method According to the sequences of Tor2 M gene published on GenBank, we designed a pair of primers for the amplification of the M gene fragment from the isolate of SARS-CoV GD322 by the method of RT-PCR. The amplified M fragment was cloned into expression vetor pET-32(a), and sequenced. The predicted amino acid sequence of M protein of SARS-CoV GD322 isolate and those of 81 isolates were analyzed by DNAstar and Clustal X. Result The sequence results showed the homology of the M gene of GD322 and Tor2 was 99.86%. When compared with the M proteins of the 81 isolates, the sequence is identical with the M protein of 41 isolates (50.62%); shows only one amino acid difference (99.55% homology) with 37 isolates (45.68%); shows two amino acids difference (99.10% homology) with 3 isolates. Conclusion The M gene of SARS-CoV GD322 isolate was cloned, and the M protein of the SARS-CoV is highly conserved.