过敏原肌质钙结合蛋白在毕赤酵母中的表达、优化及免疫反应性研究

目的 探究天然肌质钙结合蛋白(sarcoplasmic calcium binding protein, SCP)的可替代物,为蟹类过敏原的检测提供基础材料,本研究首次利用毕赤酵母(Pichia pastoris, P. pastoris)高效表达表达三疣梭子蟹(Portunus trituberculatus)重要过敏原SCP,并检验其免疫反应性。方法 根据毕赤酵母的密码子偏好性优化SCP基因并构建重组质粒。将其热激转化至P. pastoris GS115菌株后经遗传霉素(Geneticin, G418)筛选获得阳性高拷贝子。最后通过甲醇诱导表达重组SCP并结合免疫印记(Western blotting, WB)和间接酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)验证其免疫反应性。结果 SCP在P. pastoris GS115中实现了可溶性高效表达,其表观分子量约为28 kDa。在摇瓶水平下,最佳诱导条件为pH为6.0、每24 h添加1.0%(v/v)甲醇,于28℃发酵144 h,在此条件下,纯度为91.6%的SCP产量可达15 mg/L。WB和间接ELISA结果表明,重组SCP具有IgG结合能力。结论 毕赤酵母表达系统可以得到纯度较高且免疫反应性良好的重组SCP。本研究为SCP的理化研究及产业化应用奠定了基础,并有望促进特异性甲壳类过敏原检测的发展。

Expression, optimization, and immunoreactivity of an allergen sarcoplasmic calcium binding protein in Pichia pastoris

Objective To explore substitutes for the natural sarcoplasmic calcium binding protein (SCP) as a basis for detection of crab allergies. For the first time, this study utilized the efficient expression of SCP, an important allergen in Portunus trituberculatus, using the Pichia pastoris (P. pastoris) system and examined its immunoreactivity. Methods The SCP gene was optimized based on the codon preference of P. pastoris, and a recombinant plasmid was constructed. After heat shock transformation into the P. pastoris GS115 strain, positive high-copy transformants were selected using geneticin (G418). Recombinant SCP was then induced by methanol, and its immunoreactivity was validated using Western blotting (WB) and enzyme-linked immunosorbent assays (ELISA). Results Soluble and efficient expression of SCP was achieved in P. pastoris GS115, with an apparent molecular weight of approximately 28 kDa. At the flask level, the optimal induction conditions were pH 6.0 and the addition of 1.0% (v/v) methanol every 24 hours, with fermentation at 28°C for 144 hours. Under these conditions, the yield of purified SCP with a purity of 91.6% reached 15 mg/L. WB and indirect ELISA results demonstrated the IgG binding ability of the recombinant SCP. Conclusion The P. pastoris expression system yielded highly pure and immunoreactive recombinant SCP. This study laid the foundation for the physicochemical research and industrial application of SCP and is expected to promote the development of specific crustacean allergen detection.