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目的克隆SARS冠状病毒M蛋白基因编码序列,构建原核重组表达质粒pET23a-M,并在大肠杆菌BL21(DE3)中进行表达、纯化。表达产物用Western Blot检测其抗原性。方法RT-PCR扩增M编码基因目的片段,胶回收纯化,插入克隆载体pMD-18T载体并转化大肠杆菌DH5α。序列测定后亚克隆至表达质粒载体pET23a,构建重组体pET23a-M,转化大肠杆菌DH5α。筛选阳性克隆,以限制性酶切分析鉴定后,转化大肠杆菌BL21(DE3)以异丙基硫代半乳糖苷(IPTG)进行诱导表达。用SDS-PAGE与免疫印迹分析表达产物。结果PCR扩增出约675bp的特异性片段,与预期片段大小相符,测序鉴定无有义突变;所构建的pET23a-M重组体阳性克隆经PCR与双酶切鉴定,与预期结果一致;SDS-PAGE显示表达产物约27kD;表达产物经金属螯和层析纯化;免疫印迹表明表达产物能被混合的SARS病人恢复期血清识别。结论成功克隆了SARS冠状病毒M蛋白基因编码序列,构建了pET23a-M表达质粒,诱导表达并纯化出了SARS冠状病毒M蛋白,并以免疫印迹鉴定。本研究的成功为进一步进行SARS病毒诊断试剂与疫苗的开发奠定了基础。
Objective To clone the coding sequence of SARS-CoV M gene and construct prokaryotic recombinant plasmid pET23a-M. The recombinant plasmid was expressed in E. coli BL21 (DE3) and purified. The expression product was detected by Western Blot antigenicity. Methods The target fragment of M gene was amplified by RT-PCR, purified by gel filtration and inserted into the cloning vector pMD-18T and transformed into E. coli DH5α. After sequencing, the recombinant plasmid pET23a-M was subcloned into expression plasmid pET23a-M and transformed into E. coli DH5α. Positive clones were screened and identified by restriction analysis. The E.coli BL21 (DE3) was transformed into E.coli BL21 (DE3) and induced with isopropylthiogalactoside (IPTG). The expression product was analyzed by SDS-PAGE and Western blotting. Results The specific fragment of 675bp was amplified by PCR, which was consistent with the size of the expected fragment. The recombinant plasmid pET23a-M was identified by PCR and double enzyme digestion. The result of SDS- PAGE showed about 27kD of expression product; the expression product was purified by metal chelation and chromatography; Western blotting showed that the expressed product could be recognized by convalescent serum of mixed SARS patients. Conclusion The coding sequence of M protein gene of SARS coronavirus was successfully cloned. The pET23a-M expression plasmid was constructed, and the M protein of SARS-CoV was induced and purified, and identified by Western blot. The success of this study lays the foundation for the further development of SARS virus diagnostic reagents and vaccines.