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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 B (RTB) chain protein and lay the foundation for the preparation of RTB-specific antibodies. Methods: RTB coding sequence was optimized by codon optimized software. The RTB gene fragment was amplified by two-step overlap PCR using 18 long oligonucleotide primers and 6 × H is tagged at the C terminus of RTB. The expression vector pBV220 was inserted into E.coli DH5α to obtain the expression engineering strain, and the expression conditions and the purification conditions were optimized. The antigenicity of recombinant RTB protein was identified by Western blot and indirect ELISA. Results: The recombinant E. coli DH5α was expressed in inclusion bodies at 42 ℃ for 3 h, accounting for 65.48% of the total bacterial proteins. SDS-PAGE analysis showed that the expression of protein bands and RTB relative molecular mass Consistent with about 29 × 103. The expressed product was purified by N i-NTA affinity chromatography with the purity of 96.21% and about 25 mg / L recombinant RTB protein. CONCLUSION: RTB gene was synthesized by overlapping PCR in China for the first time and high expression was achieved. The purified recombinant RTB protein was recognized by anti-natural ricin multi-antibody, which laid the foundation for the preparation of specific antibody and establishment of rapid detection method.