[关键词]
[摘要]
本文采用硫酸铵沉淀法,分别以生理盐水,蒸馏水,pH=7.2的Tris-HCl缓冲液,pH=7.2的磷酸缓冲液为浸提液,对西兰苔凝集素进行提取,然后依次利用DEAE-52阴离子交换层析和SephadexG-75对凝集素粗品进行分离纯化,并对纯化后的西兰苔凝集素的部分性质进行研究。结果表明:pH=7.2的Tris-HCl缓冲液的提取率最高,为3.57±0.02%;纯化后的西兰苔凝集素分子量约为27 ku,糖含量为7.60±0.03%,其凝血活力为28;糖抑制试验表明,西兰苔凝集素的凝集活性受D-甘露糖和阿拉伯糖的抑制;热稳定性实验表明90 ℃以下凝集素的凝集活性不受温度影响,有较好的热稳定性。此外,西兰苔凝集素的最适pH范围是7~8,强酸强碱环境中其凝血活性明显下降。在含有Ca2+的溶液中其凝集活性有所增强,表明西兰苔凝集素活性受Ca2+影响较大。
[Key word]
[Abstract]
Broccolini lectin (BL) was extracted from Broccolini with normal saline, distilled water, Tris-HCl buffer and phosphate buffered saline (PBS), followed by ammonium sulphate precipitation, and finally purified by chromatography DEAE-52 and Sephadex G-75. Partial characterization of BL was also studied. The results showed that Tris-HCl buffer had the highest extraction rate of 3.57±0.02%. Molecular mass of BL was estimated as 27 ku by SDS-PAGE. The BL contained 7.60±0.03% sugar and its clotting activity was 28. The sugar inhibition test showed that its hemagglutinating activity of erythrocyte could be inhibited by D- mannose and arabinose. Moreover, the lectin remained stable at 20~90 ℃ and its optimum pH was 7~8. Its clotting activity was significantly decreased in strong acid?and?strong alkali environment. The lectin reaction was also influenced by divalent cations Ca2+, in which its hemagglutinating activity was increased.
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[基金项目]
广州市基础研究项目(2012J4100123)