奶花芸豆中凝集素的提取、纯化及鉴定

Extraction,Purification and Identification of Lectin from Phaseolus vulgaris

摘要: 以新疆奶花芸豆为原料提取凝集素,以单因素实验结果为依据,结合响应面实验对提取工艺进行优化。通过硫酸铵分级沉淀、Sephadex G-50和DEAE-A50阴离子交换层析进一步纯化,并利用SDS-PAGE电泳、质谱/质谱联用(LC-MS/MS)技术进行蛋白鉴定。结果表明,最佳提取工艺为:料液比1:13 g/mL,pH7.4,提取时间18.5 h,在此条件下凝集素特异性活力为(13.26±0.26)×10³ HU/mg。经过纯化后的目标蛋白特异性活力达到(59.93±0.31)×10³ HU/mg。经鉴定目标蛋白为奶花芸豆凝集素,分子量在31 kDa左右。

Abstract: The Phaseolus vulgarisl was used as the raw material to extract lectin,based on the single factor experiment,the extraction process was optimized by response surface experiment. Samples were further purified by ammonium sulfate fractionation,Sephadex G-50 and DEAE-A50 anion exchange chromatography,the purified protein was identified by SDS-PAGE electrophoresis and LC-MS/MS. Results showed that,the optimum extraction conditions were determined as the ratio of material to liquid 1∶13 g/mL,pH7.4 and extraction time 18.5 h the hemagglutinating activity of lectin was(13.26±0.26)×10³ HU/mg.The hemagglutinating activity of the purified target protein reached(59.93±0.31)×10³ HU/mg,the target protein was determined as lectin and the molecular weight was around 31 kDa.