SARS冠状病毒Mc区克隆及序列分析

目的：构建SARS冠状病毒（SARS-CoV）GD322株M基因膜内区（Mc区）重组表达质粒，并对目的序列进行序列分析。方法：根据GenBank中公布的SARS-CoV Tor2株M基因序列，设计一对引物，采用RT-PCR法从SARS-CoV GD322株基因组中扩增Mc区．克隆至pET-32a（＋）载体中，转化大肠杆菌BL21后测序，利用Clustal X分析所测序列翻译的氨基酸与81株SARS-CoVMc区翻译的氨基酸序列的差异。结果：测序结果表明该区与Tor2株对应核苷酸序列同源性为100％。与已收集的81株SARS-CoVMc区所译氨基酸（Mc蛋白）相比，与77株（占95．1％）Mc蛋白完全同源．与4株（占4．9％）仅有一个氨基酸改变。结论：本试验成功构建SARS-CoV Mc区重组表达质粒：SARS-CoV各毒株间Mc蛋白氨基酸序列差异极小，提示利用任一毒株Mc蛋白制备单抗和疫苗具有普遍意义．

Cloning and sequence analysis of SARS-CoV Mc region

Objective To construct a recombinant expressing plasmid of SARS-CoV GD322 Mc region and analyze its sequences. Methods Based on the sequences of Tor2 M gene published on GenBank, we generated a pair of primers, amplified Mc fragments from the isolate of SARS-CoV GD322 using RT-PCR, then inserted the Mc fragments into expression vetor pET-32a( ), and then transformed E. coli BL21 with the chimeric plasmid, finally sequenced the products. The amino acid sequences of Mc protein from the SARS-CoV GD322 isolate and those from 81 SARS-CoV isolates were analyzed by Clustal X. Results The sequencing result showed the nucleotide sequence in GD322 had a homology of 100% with the corresponding sequence in Tor2;The amino acid sequence of Mc protein translated by GD322 Mc region was completely homologous to that of 77 out of the 81 SARS-CoV isolates collected from GenBank and only had one different amino acid as compared with the remaining 4 isolates. Conclusions The recombinant expression plasmid of SARS-CoV Mc region is successfully constructed. There is little difference in the amino acid sequence of Mc protein among the isolates, suggesting the monocloned antibody or the vaccine for SARS-CoV generated from the Mc proteins of any isolate has a universal significance.