中药白芨提取物作为基因载体的制备与表征

 目的分离纯化并制备阳离子型白芨多糖,研究其表征,探讨其作为基因载体的可能性。方法经热水抽提,乙醇沉淀,Sevag法脱蛋白,阴离子交换纤维素柱(DE-52)和凝胶柱(Sephadex G-100)色谱,分离纯化得白芨多糖,通过高碘酸钾氧化得到含醛基的还原性多糖,该还原性多糖与精胺在碱性条件下形成希夫碱式共轭物,经硼氢化钠还原得含伯胺基的阳离子型多糖,分别通过盐酸羟胺法和三硝基苯磺酸法测醛基与伯胺基的产量,并考察了氧化反应时间对反应产物的影响。进一步通过琼脂糖凝胶电泳检测该阳离子型多糖对基因的结合与保护作用。结果所提纯的白芨多糖为一种不含阳离子基团的非还原性多糖,通过胺化还原反应的方法可成功制备出含伯胺基的阳离子型多糖,红外光谱证实了多糖上伯胺基的存在,进一步的研究表明,该阳离子型多糖可以结合并保护质粒DNA。结论通过胺化还原反应方法制备的阳离子型白芨多糖,有望作为一种多糖基的多聚阳离子型基因载体在真核细胞基因转染中发挥作用。

Preparation and Characterization of a Non-viral Gene Vector from a Polysaccharide Isolated from the Chinese Medicinal Herb Bletilla striata

OBJECTIVE To prepare the cationic product of a polysaccharide isolated from Chinese medicinal herb Bletilla striata,and to investigate the characterization and the possibility of being a non-viral gene vector.METHODS Bletilla striata polysaccharide was prepared by ethanol precipitation followed the hot water extraction Bletilla striata stem and deproteinized with Sevag method.It was further isolated by ion-exchange chromatography on DE-52 column and gel filtration on Sephadex G-100 column.Cationic polysaccharide was synthesized by conjugation of spermine to oxidized polysaccharide.The ability of the caionic polysaccharide to incorporate and protect plasmid DNA was measured.RESULTS There is no cationoid primary amine and reductive carbonyl in the polysaccharide isolated from the Chinese medicinal herb Bletilla striata.The structure of cationic Bletilla striata polysaccharide contained primary amine group was confirmed by FT-IR.The synthesized cationic product incorporated and protectd plasmid DNA.CONCLUSION The cationic Bletilla striata polysaccharide synthesized by grafted spermine to the polysaccharide isolated from the Chinese medicinal herb Bletilla striata could incorporate and protect plasmid DNA,which would be an ideal non-viral gene vector in gene transduction.