Prokaryotic Expression of RaDtxR Gene of Riemerella anatipestifer
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摘要: 本研究根据前期研究获得的鸭疫里默氏菌铁依赖抑制子（RaDtxR）基因序列特征设计特异性引物，并在引物上下游引入BamHⅠ和Xho Ⅰ酶切位点，将其克隆到原核表达载体pGEX-4T-1上，构建原核表达重组质粒pGEX-4T-1-RaDtxR，对其进行IPTG诱导表达条件优化和表达目的蛋白的生物学活性鉴定。SDS-PAGE和Western-blot分析表明，分子量约为51 ku目的蛋白得到表达，并具有良好的生物学活性。本研究成功对鸭疫里默氏菌RaDtxR基因进行表达，为研究鸭疫里默氏菌RaDtxR蛋白生物学功能奠定基础。Abstract: Based on the previously obtained characterization on the RaDtxR gene of Riemerella anatipestifer, upstream and downstream primers were designed with BamHⅠ and Xho Ⅰ restriction enzyme sites added at the 5'-teriminal. These quence of the RaDtxR gene was amplified and cloned into the prokaryotic expression vector pGEX-4T-1 to generate recombinant plasmids pGEX-4T-1-RaDtxR. Then, the expression conditions were optimized by IPTG induced by SDS-PAGE, and the biological activity identified by Western blot. SDS-PAGE and Western blot confirmed that the protein with a molecular weight approximating 51 ku was well expressed with a significant biological activity. Therefore, the highly efficient expression ofthe RaDtxR protein would provide a basis for further study associated with the specificgene.
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Key words:
- Riemerella anatipestifer /
- RaDtxR gene /
- prokaryotic expression
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图 1 RaDtxR基因的PCR扩增
注：M为DL 2 000 Marker；1为目的片段；2为阴性对照。
Figure 1. PCR amplification of RaDtxR gene
图 2 重组质粒的双酶切鉴定
注：1为pGEX-4T-1空载体；2为阳性重组表达质粒pGEX-4T-1-RaDtxR；M为DL 15 000 Marker。
Figure 2. Double digestion analysis on recombinant plasmids
图 3 表达蛋白的SDS-PAGE分析
注：1~5为诱导时间 (1、2、3、4、5h)；6为pGEX-4T-1空载体诱导；M为蛋白质标准。
Figure 3. SDS-PAGE analysis on expressed proteins
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