禽蛋黄过敏原Gal d 6的重组表达、纯化鉴定
Recombinant Expression, Purification and Identification of Egg Yolk Allergen Gal d 6
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摘要: 目的:建立表达蛋黄Gal d 6 重组蛋白工程菌,并鉴定重组Gal d6蛋白免疫活性。方法:直接合成Gal d 6 DNA,与pET-28a构建重组质粒,转化E.coli BL21经IPTG诱导表达;优化表达条件制备重组Gal d 6蛋白,经镍离子亲和层析柱纯化得到纯品蛋白;纯化Gal d6蛋白经间接ELISA及western blot鉴定Gal d 6蛋白免疫活性。结果:经DNA测序、SDS-PAGE、禽蛋过敏血清鉴定表达Gal d 6蛋白具有免疫活性,经镍柱亲和层析获得单一条带。该工程菌最佳表达条件为37 ℃,0.5 mmol/L IPTG,2 h,收获量每升菌液可收获重组蛋白约9.1 mg。重组蛋白与禽蛋过敏血清具有良好反应性。结论:建立稳定表达具有免疫活性的Gal d 6蛋白的工程菌株。Abstract: Objective:To establish an egg yolk Gal d 6 expressed engineering strain and identify the immunological activity of rGal d 6 protein. Methods:The Gal d 6 gene was directly synthesized, and then cloned into the vector pET-28a. The constructed recombinant plasmid was transformed into E.coli BL21 and the protein was induced expression by IPTG and purified by Ni-NTA.Identification of rGal d 6 immunoreactivity was detected by indirected ELISA and western blot. Results:The optimum expression conditions for the engineered bacteria were 37℃, 0.5 mmol/L IPTG, 2 hours, and the yield could reach 9.1 mg per liter. A single band was obtained by Ni-NTA. The recombinant protein had good reactivity with egg allergy serum. Conclusion:An engineered strain with stable expression of Gal d 6 protein with immunoreactivity was established.
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