重组相思子毒素B链蛋白的制备与分析  

Construction and characterization of a recombinant abrin B chain protein

作　　者：王俊虹[1] 杨帆[3] 魏茂提[1,2] 

机构地区：[1]天津市职业与环境危害防制重点实验室,武警后勤学院救援医学系部队卫生统计和流行病学教研室,300309 [2]天津市职业与环境危害防制重点实验室,武警后勤学院军事心理学教研室,300309 [3]不详

出　　处：《免疫学杂志》2016年第2期164-167,共4页Immunological Journal

基　　金：军事预防医学“2110”三期工程建设项目

摘　　要：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。This experiment designed to construct, express and purify a recombinant abrin B chain protein （rATB）, and then assess its antigenicity and toxicity, rATB was prepared by replacing rare codon with synonymous codons of high-frequency and subcloned to construct recombinant expression vector pQE80L-ATB. After expression and purification, rATB was determined by immunoblotting and MTS for its antigenicity and toxicity. Data showed that an 804 bp gene encoding the recombinant protein with 268 amino acid residues was acquired. M15/ pQESOL-ATB was induced at 30℃ by 0.1 mmol/L IPTG for 5 h, and the relative mass of ATB was approximate 30 kDa. SDS-PAGE indicated that the purity of the protein was up to 97% after Ni-NTA purification; Western blot indicated that rATB could react specifically with the rabbit pAb against AT; MTS showed that rATB was non-toxic even at high doses. In conclusion, rATB from E. coli possess good antigenicity and can be highly expressed, which proved a base for developing effective and non-toxic vaccine.

关 键 词：相思子毒素B链 抗原性 生物恐怖 

分 类 号：R392-33[医药卫生—免疫学]