论文部分内容阅读
目的:构建截短的泡球蚴18(Em18)基因的原核表达质粒,获得高效表达、有生物活性的重组蛋白,并对其进行初步鉴定。方法:DNAman软件设计引物,PCR法扩增并构建截短的pET41a-Em18．1原核表达质粒,测序鉴定插入序列正确性。异丙基硫代-β-D-半乳糖苷(IPTG)诱导、表达和纯化rEm18．1-GST重组蛋白,采用SDS-PAGE 电泳及Western免疫印迹法对其进行初步鉴定。结果:成功构建出截短的pET41a-Em18．1,SDS-PAGE检测表明rEm18．1-GST重组蛋白得到成功表达,在相对分子量为41 kDa处有表达条带。Western blot结果显示 rEm18．1-GST重组蛋白能被AE病人阳性血清识别。结论:截短的pET41a-Em18．1原核表达质粒表达的 rEm18．1-GST重组蛋白具有良好的抗原性,为Em18抗原表位的分析奠定基础。
OBJECTIVE: To construct a prokaryotic expression plasmid of truncated Em18 gene and to obtain a recombinant protein with high expression and biological activity, and preliminary identification of the recombinant protein. Methods: DNAman software was used to design the primers. The truncated pET41a-Em18.1 prokaryotic expression plasmid was amplified by PCR and sequenced to confirm the correctness of the inserted sequence. The rEm18.1-GST recombinant protein was induced, expressed and purified by isopropylthio-β-D-galactoside (IPTG). The recombinant protein was identified by SDS-PAGE electrophoresis and Western blotting. Results: The truncated pET41a-Em18.1 was successfully constructed. SDS-PAGE analysis showed that the rEm18.1-GST recombinant protein was successfully expressed and expressed at a relative molecular mass of 41 kDa. Western blot results showed that rEm18.1-GST recombinant protein can be recognized by AE positive serum. CONCLUSION: The rEm18.1-GST recombinant protein expressed in the truncated pET41a-Em18.1 prokaryotic expression plasmid has good antigenicity and lays the foundation for the analysis of the epitope of Em18.