灰树花中一种抗烟草花叶病毒的蛋白质的纯化及其性质

Purification and Partial Characterization of a Protein Inhibitor of Tobacco mosaic virus Infection From The Maitake (Grifola frondosa)

通过硫酸铵分级沉淀、阴离子交换柱层析及凝胶过滤柱层析 ,从灰树花子实体中分离到了一种具有抑制烟草花叶病毒 (TMV)侵染活性的热稳定蛋白———GFAP .经常规凝胶电泳和等电聚焦电泳显示为单一条带 ,而SDS 聚丙烯酰胺凝胶电泳结果证明该蛋白质含有两个亚基 ,其分子质量分别为 34ku、 4 0ku .等电聚焦测定蛋白质 pI为 3 76 ,含糖量约为 2 % .GFAP的 4 0ku条带N端氨基酸序列为ACCVPSVTEFENAINSDPVM ,将其与GenBank中的氨基酸序列检索比较后没有发现同源序列 ,预示可能为一新的氨基酸序列 .GFAP与TMV混合接种心叶烟 ,当GFAP浓度为 32mg/L时即可完全抑制浓度为 10mg/L的TMV的侵染 ,而 4mg/L的GFAP对浓度为 4 0mg/L的TMV的抑制率仍可达 6 0 %以上 .

A heat stable protein, named GFAP, was isolated and purified from the fruiting bodies of edible fungus Grifola frondosa by a procedure of (NH 4) 2SO 4 precipitation, and ion-exchange chromatography on DEAE-Sepharose column and gel filtration on Sepharose-6B column. According to PAGE and IEF,GFAP had a single band with pI 3.67. And it consisted of two subunits of 34 ku and 40 ku when encounted by SDS-PAGE. GFAP is identified as a glycoprotein containing 2% sugar. The N-terminal amino acid sequence of the 40 ku subunit is ACCVPSVTEFENAINSDPVM,which has no homology with other sequences in GenBank. GFAP possesses the inhibitory activity against the infection of Tobacco mosaic virus. When mixed with TMV(10 mg/L) and inoculated in local lesion host,Nicotiana glutinosa, GFAP was able to completely inhibit the infection of TMV,and with TMV(40 mg/L),it still had an infection effect of higher than 60% at the concentration of 4 mg/L.