重组蓖麻、相思子毒素A链嵌合突变体的制备与分析

Production and Characterization of a Recombinant Chimeric Protein Consisting Mutant Ricin A Chain and Abrin A Chain

目的:构建蓖麻毒素(RIC)、相思子毒素(ABR)A链突变体的嵌合体蛋白,实现嵌合体蛋白的可溶性表达、纯化及抗原性分析。方法:采用柔性linker连接RIC A链突变体(mRICAD75AV76MY80A)和ABR A链突变体(mABRAE164AR167L),构建嵌合体基因mRICA/mABRA,将该嵌合体基因亚克隆至原核载体pQE80L构建表达质粒pQE80L-mRICA/mABRA,再转化至大肠杆菌M15获得表达工程菌株M15/pQE80L-mRICA/mABRA,工程菌在18℃经0.1 mmol/L的IPTG诱导14 h,表达的嵌合体蛋白经Ni-NTA亲和层析柱纯化,通过ELISA和Western印迹检测嵌合体蛋白的抗原性。结果:所获得的mRICA/mABRA嵌合体基因经一致性比对分析,与预计嵌合基因的序列一致性为100%,其开放读框全长1572 bp,编码524个氨基酸残基;重组表达质粒pQE80L-mRICA/mABRA经PCR及双酶切鉴定证明构建正确,嵌合体蛋白相对分子质量约为62×103,与预测相符,可溶性的嵌合体蛋白经Ni-NTA亲和层析柱纯化,纯度可达99%;间接ELISA和Western印迹结果表明,嵌合体蛋白能同时与抗RIC多克隆抗体和抗ABR多克隆抗体发生特异的抗原抗体反应。结论:得到的mRICA/mABRA嵌合体蛋白具有良好的抗原性,为研制新型RIC和ABR双价疫苗奠定了重要基础。

Objective： To construct the chimeric protein mRICA/mABRA consisting mutant ricin（RIC） A chain and abrin（ABR） A chain,express it in the cytoplasm of Escherichia coli M15,and evaluate its antigenicity.Methods： The chimeric gene mRICA/mABRA was produced by connecting the genes of mutant ricin A chain（mRICAD75AV76MY80A） and mutant abrin A chain（mABRAE164AR167L） via linker.The recombinant expression vector pQE80L-mRICA/mABRA was transformed into E.coli M15 for expression under induction of IPTG at 18℃.The product was purified using chelating nickel-nitrilotriacetic acid（Ni-NTA） affinity chromatography,and then its antigenicity was determined by ELISA and Western blot.Results： Both PCR and sequencing analysis proved that recombinant plasmid pQE80L-mRICA/mABRA was produced correctly,the sequence of chimeric gene mRICA/mABRA had a 100% homology with the sequence of designed gene,with an open reading frame of 1572 bp,which could encode the chimeric protein mRICA/mABRA with 524 amino acid residues.The Mr of soluble expressed protein mRICA/mABRA was approximately 62×103.The protein reached a purity of about 99% after purification,and showed specific affinity with both polyclonal antibodies against native ricin and abrin.Conclusion： This study describes the generation of a substantial amount of chimeric protein mRICA/mABRA from E.coli and the potential application of mRICA/mABRA as an effective chimeric vaccine candidate that can protect against ricin and abrin simultaneously.