猪乳中一高分子量蛋白质的分离纯化和鉴定

摘要

对猪乳中一高分子量蛋白(HMWP)进行了分离纯化,并对其某些生化性质进行了鉴定.猪乳通过去脂得到脱脂乳,再去除酪蛋白得到乳清.对乳清进行硫酸铵分级盐析,猪乳中HMWP在40%饱和度硫酸铵盐析下有最大沉淀.收集40 %饱和度硫酸铵盐析沉淀,经过溶解、透析得到HMWP的粗品.通过Mono Q离子交换柱,对其粗品进行两次层析提纯,得到了HMWP纯品,其纯度和得率分别为97.85 %和12.31 %.多种植物凝集素的Western blotting鉴定表明,HMWP是一个糖基种类较少的糖蛋白,含有Man和GlcNAc.SDS-PAGE和凝胶过滤分别测得HMWP的分子量为114.8 kD和115.0 kD.通过等电点测定,HMWP的pI为5.10.HMWP的氨基酸组分分析得知,其富含Asp、Glu、Gly和Cys,疏水性氨基酸较低,仅占15.59 %摩尔分数.这些结果说明HMWP是一个易溶于水的、酸性的分泌性单体球蛋白.N端氨基酸序列测定结果为Ala-Leu-Val-Gln-Ser-Gly-Leu-Asn-Leu-Val,通过从网络Genbank 检索没有发现其同源蛋白的序列,说明其可能是一个新蛋白.%A high molecular weight protein (HMWP) was purified from sow milk, its ceitain biochemical characteristics were identified. The sow milk was centrifuged to remove the cream and cellular debris, the skim milk was obtained. Whey was made after removal of caseins, and then fractionated by different saturation (NH4)2SO4. The resulting protein pellets of 40 % saturation (NH4)2SO4 were resuspended in distilled water and dialyzed against distilled water to obtain crude sample. The crude sample was chromatographed by Mono Q column, further purified by Mono Q column again. Finally, the purified HMWP was obtained, which exhibited a single band on SDS-PAGE in reduction. The purity and yield of the purified HMWP reached above 97.85 % and 12.31 % respectively. By SDS-PAGE and gel filtration, the molecular weight of HMWP was estimated to be about 114.8 kD and 115.0 kD respectively. Identified by western blotting of lectin, HMWP was a kind of glycoprotein with less kinds of sugar, containing mannose (Man) and N-acetylglucosamine (GlcNAc). By isoelectric focusing analysis, HMWP pI was found to be 5.1. The result of amino acid assay showed that HMWP was abundant in glutamate, cysteine, glycine, aspartic acid and proline. The content of hydrophobic amino acids was lower, only taking up 15.59 mol% of total amino acids assayed. These results suggested that HMWP may be a soluble, acidic, secretary and monomeric globulin. The first 10 N-terminal amino acid sequence of HMWP was Ala-Leu-Val-Gln-Ser-Gly-Leu-Asn-Leu-Val. The sequence was blasted against GenBank, no protein known showed significant similarity with this sequence, which demonstrated that HMWP could be a novel one.