Expression ofσC gene of muscovy duck reovirus,MW9710 strain,by using pMAL-p2X system
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摘要: 根据Genbank中MDRV 89026毒株S4基因序列设计引物,通过RT-PCR获得番鸭呼肠孤病毒MW9710株σC蛋白基因,将此σC蛋白基因插入原核表达载体pMAL-p2X中,得到重组表达质粒(pMAL-p2X-σC)并转化大肠杆菌BL21(DE3)中,经IPTG诱导后能高效表达,SDS-PAGE和Western-blot检测结果发现表达的σC蛋白分子质量约为72 kDa,并以可溶性蛋白和包涵体2种形式同时存在,且具有良好的反应原性。原核表达的可溶性σC蛋白为进一步开展抗原表位和诊断试剂盒研究奠定了基础。Abstract: To obtain recombinant Muscovy duck reovirus(MDRV) _σC protein by prokaryotic expression for further study on its functions,the encoding sequence of the _σC gene from the MDRV MW9710 strain was amplified with RT-PCR and inserted into pMAL-p2X vector to establish the prokaryotic expression plasmid.After transforming the plasmid,pMAL-p2X-_σC,into E.coil BL21(DE3),the bacteria were induced by IPTG.The protein was expressed efficiently in both soluble and insoluble forms,and was positively recognized by the reference serum.The SDS-PAGE and western-blot analysis showed the recombinant pMAL-p2X-_σC produced had an apparent molecular weight of 72KDa.The soluble _σC protein was purified and obtained using MBP-affinity chromatography.The result provided a foundation for the gene’s epitope identification and development of a clinical diagnostic kit in the future.
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Key words:
- Muscovy duck revoirus /
- σC protein /
- pMAL-p2X /
- prokaryotic expression
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