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目的构建、表达和纯化相思子毒素B(ATB)链蛋白并分析其抗原性和毒性。方法运用密码子优化软件优化ATB基因,合成目的基因亚克隆至原核载体p QE-80L构建表达质粒p QE80L-ATB,转化至E.coli M15获得表达工程菌株,并对诱导表达条件和纯化条件进行优化;通过Western blot检测重组蛋白的抗原性;采用人正常肺上皮细胞Beas-2B和胃黏膜细胞GES-1进行重组蛋白的细胞毒性实验。结果 ATB开放阅读框架全长804 bp,编码268个氨基酸残基;表达工程菌株在30℃、1 mmol/L IPTG,3 h后获得包涵体形式表达的目的蛋白,相对分子质量均约为30 000,经Ni-NTA亲和层析柱纯化后纯度达97%以上,Western blot和MTS实验结果表明,目的蛋白能特异性识别抗相思子毒素多克隆抗体且无毒性。结论重组ATB蛋白实现高表达,具有良好的抗原性,为相关疫苗研究奠定基础。
Objective To construct, express and purify Aciton Toxin B (ATB) chain protein and analyze its antigenicity and toxicity. Methods The ATB gene was optimized by codon optimized software. The target gene was subcloned into prokaryotic vector pQE-80L to construct expression plasmid pQE80L-ATB. The recombinant plasmid was transformed into E.coli M15 to obtain the expression engineering strain. The induced expression conditions and purification conditions The recombinant protein was tested for its antigenicity by Western blot. The cytotoxicity of recombinant protein was tested using human normal epithelial cells Beas-2B and gastric mucosal cells GES-1. Results The ATB open reading frame (ORF) was 804 bp in length and encoded 268 amino acid residues. The target protein was expressed in the inclusion bodies after the recombinant strain was expressed at 30 ℃ and 1 mmol / L IPTG for 3 h. The relative molecular mass of the expressed protein was about 30 000 , Purified by Ni-NTA affinity chromatography, the purity of more than 97%, Western blot and MTS experimental results show that the target protein can specifically identify the anti-acteins polyclonal antibodies and non-toxic. Conclusion The recombinant ATB protein is highly expressed and has good antigenicity, which lays the foundation for the research of related vaccines.