重叠PCR合成蓖麻毒素B链基因及其在大肠杆菌中表达和抗原性分析

Expression of recombinant ricin toxin B chain protein in Escherichia coli from an overlapping PCR-synthesized gene and its antigenicity

目的:表达蓖麻毒素B(RTB)链蛋白,为制备RTB特异性抗体奠定基础。方法:采用密码子优化软件对RTB编码基因序列重新优化设计,以18条长片段寡核苷酸引物经两步重叠PCR合成RTB基因片段,并在RTB的C端引入6×H is标签序列,插入表达载体pBV220,转化E.coliDH5α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。利用W estern印迹和间接ELISA方法鉴定重组RTB蛋白的抗原性。结果:表达工程菌株E.coliDH5α在42℃经诱导3 h后获得包涵体形式表达的目的蛋白,约占菌体总蛋白的65.48%,SDS-PAGE分析显示表达的蛋白区带与RTB相对分子质量相符,为29×103左右。表达产物经N i-NTA亲和层析法一步纯化,蛋白纯度达96.21%,并可得到约25 mg/L重组RTB蛋白。结论:在国内首次应用重叠PCR合成RTB基因并实现高表达,纯化重组RTB蛋白可被抗天然蓖麻毒素多抗所识别,为制备特异性抗体及建立快速检测方法打下了基础。

Objective： To express ricin toxin B（RTB） chain protein and prepare the antibody against RTB. Methods： The optimum design of 786 bp gene fragment was carried out by replacing rare x codons with high-frequency ones, and the 786 bp fragment was synthesized using overlapping PCR by two steps. After confirmed by sequencing,6 × His-tagged gene sequences were added to the C-termini of RTB gene fragments, which were subcloned to construct recombinant expression vector pBV220-RTB. Then it was transformed into E. coli DH5α. The expression and purification condition was optimized. The antigenicity of rRTB was identified by Western blot and indirect ELISA. Results： Recombinant strain E. coli DH5α pBV220-RTB was induced at 42℃ for 3 hours. A specific expression band with a relative molecular mass 29× 10^3 was detected by SDS-PAGE. Torget protein existed in form of inclusion and amounted to 65.48% of total protein. The purity of expressed protein purified by one-step Ni-NTA affinity chromatography could reach 96.21%, and 25 mg/L of recombinant protein was obtained. Both Western blot and indirect ELISA showed that the antiserum against native ricin had a specific affinity for the recombinant RTB protein. Conclusions： It is the first time to synthesize RTB gene using overlapping PCR, and recombinant protein is expressed successfully in E. coli DH5α cells. The purified recombinant RTB protein can be detected by muhiclonal antibody against native ricin. This lays foundation for preparing specific antibodies against RTB and detecting ricin toxin.