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提取芹菜总RNA,反转录PCR(RT-PCR)扩增出过敏原蛋白Apig1.02基因,经PCR、酶切和序列测定后,得到重组质粒pET-32a-Apig1.02。将重组质粒转化到BL21(DE3)pLys感受态细胞中,用1mmol/L异丙基硫代-B-D-半乳糖苷(IPTG)对转化菌进行诱导表达及SDS-PAGE分析后,将表达蛋白提取纯化,制备单克隆抗体并进行Western blot分析。结果表明芹菜过敏原蛋白Apig1.02在体外获得了高效表达,表达融合蛋白分子质量约35ku,诱导6h后表达量最高,占菌体总蛋白的40%左右;制备的单克隆抗体能与表达蛋白发生免疫印迹反应,说明所表达的蛋白具有良好的免疫原性。该研究结果为建立芹菜过敏原蛋白的免疫学检测方法奠定了基础。
The total RNA of celery was extracted and the allergen Apig1.02 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The recombinant plasmid pET-32a-Apig1.02 was obtained after PCR, restriction enzyme digestion and sequencing. The recombinant plasmids were transformed into BL21 (DE3) pLys competent cells. The transformed bacteria were induced with 1 mmol / L IPTG and analyzed by SDS-PAGE. The expressed proteins were extracted Purified, prepared monoclonal antibodies and Western blot analysis. The results showed that celery allergen protein Apig1.02 was highly expressed in vitro. The molecular weight of the expressed fusion protein was about 35ku. After 6 hours of induction, the expression level of Apig1.02 protein was about 40% of the total bacterial protein. The prepared monoclonal antibody could react with the expressed protein Immunoblotting reaction occurred, indicating that the expressed protein has good immunogenicity. The results of this study laid the foundation for establishing an immunological detection method for celery allergen protein.