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目的表达获得较纯的细粒棘球绦虫AgB8/3重组抗原(rEgAgB8/3)。方法根据GeneBank登陆号(AF362442)下载目的基因核酸序列,利用DNAman软件设计引物,对EgAgB8/3编码分泌型多肽片段的核酸序列进行PCR扩增,测序鉴定其正确性后定向连入原核表达质粒pTWIN1上,并转化至大肠杆菌ER2566,IPTG诱导表达CBD-intein1-EgAgB8/3融合蛋白后进行纯化,用SDS-PAGE电泳和western blot分析鉴定融合蛋白与目的蛋白rEgAgB8/3的表达量和纯度。结果成功克隆获得EgAgB8/3基因目的片段和具有蛋白自剪切功能的重组表达载体pTWIN1-EgAgB8/3,并鉴定其表达的融合蛋白主要以可溶形式存在;构建的重组载体中融合标签的几丁质结合域(CBD)和内含肽(intein1)分别使亲和层析纯化和融合标签自剪切一步完成。结论成功的构建重组表达载体pTWIN1-EgAgB8/3,获得高表达融合蛋白CBD-intein1-EgAgB8/3,并经简单后续处理即可获得含极少任何额外氨基酸的可溶性目的蛋白rEgAgB8/3,尽可能保证了其原有的结构和活性,为抗rEgAgB8/3特异性单克隆抗体的快速制备奠定了基础。
Objective To obtain the more pure Echinococcus granulosus AgB8 / 3 recombinant antigen (rEgAgB8 / 3). Methods According to the GeneBank accession number (AF362442), the nucleic acid sequence of the target gene was downloaded and designed by DNAman software to amplify the nucleic acid sequence of the secreted peptide fragment of EgAgB8 / 3. The sequence was identified by sequencing and inserted into prokaryotic expression plasmid pTWIN1 The fusion protein and the target protein rEgAgB8 / 3 were identified by SDS-PAGE electrophoresis and western blot analysis. Results The target fragment of EgAgB8 / 3 gene and the recombinant expression vector pTWIN1-EgAgB8 / 3 with the function of self-shearing were successfully cloned and the fusion protein was identified to be mainly in soluble form. The constructed fusion vector Purification of the affinity chromatography and fusion tags, respectively, were done in one step from shearing, by the CBD and intein1, respectively. Conclusion The recombinant expression vector pTWIN1-EgAgB8 / 3 was successfully constructed, and the highly expressed fusion protein CBD-intein1-EgAgB8 / 3 was obtained. After a simple subsequent treatment, soluble protein of interest rEgAgB8 / 3 with very few extra amino acids could be obtained. Which ensures its original structure and activity, and lays the foundation for rapid preparation of anti-rEgAgB8 / 3 specific monoclonal antibody.