半夏凝集素的表达、纯化及抗菌性质研究
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摘要
凝集素是一种广泛存在于动植物及微生物体中的选择性结合或非共价性结合于糖基的蛋白或糖蛋白。这类蛋白的主要性质是它们可以特异性的与糖类物质作用,而且与细胞表面的糖类复合物相结合。
根据目前的生物化学、分子生物学和结构分析学的研究进展,从进化和结构相关性来分,凝集素可分成7个不同家族:葫芦科韧皮部凝集素;觅科凝集素;包含Hevein结构域的壳多糖结合蛋白;木菠罗素(jacaLin)家族;豆科凝集素;单子植物甘露糖结合凝集素;2型核糖体失活蛋白(RIP)。半夏属于天南星科,天南星科凝集素属于单子植物甘露糖结合凝集素,可以特异性的与甘露低聚糖结合。它们可以快速凝结兔血细胞,但是不能凝结人血细胞。
为了研究半夏凝集素及其亚基的性质,本研究分别构建了含半夏凝集素表达前体蛋白PTA-P、两个亚基蛋白PTA-DOM1及PTA-DOM2的原核表达载体,并成功地实现了这三个基因在E.coLi BL21中的表达了含有N端组氨酸标签的融合蛋白。表达的蛋白在镍氢离子柱上纯化并成功复性。
本研究发现融合蛋白PTA-P、PTA-DOM1和PTA-DOM2主要有以下特性:1.可以凝集兔血,但是不能凝集人血而且他们的凝血活性受到甘露糖的抑制。这些性质和天南星科其他的凝集素是相似的。2.这三种半夏凝集素蛋白对细交链孢、离蠕孢、新月弯孢的生长具有明显的抑制,而且PTA-P的抑菌活性要高于其他两种蛋白。在被测试的三种真菌中,PTA-P、PTA-DOM1和PTA-DOM2均对细交链孢显示了最高的敏感性,对新月弯孢的敏感性最低。用50μg蛋白处理真菌24小时后,发现真菌菌丝尖端发生肿大而且多生分枝。3.三种半夏凝聚素蛋白在20℃和60℃之间保持完整活性,当温度达到100℃时只保留25%的活性。4.三种半夏凝集素蛋白在pH4到pH9之间都显示了活性,在pH7.8是活性最高。研究发现三种半夏凝集素蛋白还可以抑制细菌的生长,暗示利用分子生物学手段将PTA－P及两个亚基基因导入植物使之产生抗病性可能将是一个可行的方案。
Lectins are naturally occurring proteins and glycoproteins which bind selectively and non-covalently to carbohydrate residues.They are widely distributed in living organisms including animals,plants,microorganism.The main characteristic of this class of protein is their ability to interact specifically with carbohydrates and to combine with glyco-components of the cell surface.
On the basis of recent advances in biochemistry and molecular cloning and structural analysis,virtually all currently known plant lectins can be classified into seven families according to structurally and evolutionarily-related proteins: amaranthins,Cucurbitaceae phloem lectins,lectins with hevein domain(s), jacalinrelated lectins,legume lectins,monocot mannose-binding lectins and type-2 ribosome-inactivating proteins.Pinellia ternata is a traditional Chinese medicinal plant species,belonging to the family Araceae.Araceous lectins are a group of proteins possessing at least one non-catalytic domain that binds reversibly to specific monoor oligo-saccharide(glycoconjugate).They readily agglutinated rabbit erythrocytes but were unable to agglutinate human erythrocytes irrespective of the blood group.
The property of Pinellia ternate angustate agglutinin(PTA) and its domains was investigate in this study.The pta cDNA sequence encoding for mature PTA protein and two domains were cloned into PET-28a plasmid and the resulting in pET-28a-PTA、pET-28a-DOM1 and pET-28a-DOM2 containing N-terminal His-tagged fusion protein,repectiveLy.The expressions of them in E.coli BL21 were investigated.Purification of the PTA-P、PTA-DOM1 and PTA-DOM2 recombinant proteins were performed under native conditions by nickel ion metal-affinity chromatography.
The recombinant PTA-P、PTA-DOM1 and PTA-DOM2 exhibited several characteristics:1.they exhibited hemagglutinating activity towards rabbit but not human erythrocytes,and their hemagglutinating activity could be inhibited by mannose.These profiles were similar to other Araceae lectins.2.Both PTA-P and two domains exhibited potent antitungal activity toward Alternaria altanata,Bipolaris sorokiniana and Curvularia lunata.The antifungal activities of PTA-P were obviously stronger than that of PTA-DOM1 and PTA-DOM2.Among the tested fungi, Alternaria altanata appeared the most sensitive to PTA-P with an IC50 of 0.19μM, Alternaria altanata and Bipolaris sorokiniana more sensitive than Curvularia lunata to PTA、PTA-DOM1 and PTA-DOM2.The morphological toxicity of PTA-P、PTA-DOM1 and PTA-DOM1 to Curvularia lunata was revealed by an increase in mycelial apex offshoot and tumescence after treatment with 50ug proteins for 24 h.3. The results of thermal denaturation of fusion proteins showed that the antifungal activity of PTA-P、PTA-DOM1 and PTA-DOM1 were extremely stable between 20℃and 60℃.Even heating at 100℃for 5 min caused loss of only 25%of its original activities.4.they also exhibited activity under a broad pH range from 4.0 to 9.0 with highest activity at 7.8.We also found that the three proteins were active against Escherichia coli and Stapylococcus aureus.Sum up,The PTA-P,PTA-DOM1 and PTA-DOM1 may be a promising candidate for possible applications in the genetic engineering of disease-resistant crops.
根据目前的生物化学、分子生物学和结构分析学的研究进展,从进化和结构相关性来分,凝集素可分成7个不同家族:葫芦科韧皮部凝集素;觅科凝集素;包含Hevein结构域的壳多糖结合蛋白;木菠罗素(jacaLin)家族;豆科凝集素;单子植物甘露糖结合凝集素;2型核糖体失活蛋白(RIP)。半夏属于天南星科,天南星科凝集素属于单子植物甘露糖结合凝集素,可以特异性的与甘露低聚糖结合。它们可以快速凝结兔血细胞,但是不能凝结人血细胞。
为了研究半夏凝集素及其亚基的性质,本研究分别构建了含半夏凝集素表达前体蛋白PTA-P、两个亚基蛋白PTA-DOM1及PTA-DOM2的原核表达载体,并成功地实现了这三个基因在E.coLi BL21中的表达了含有N端组氨酸标签的融合蛋白。表达的蛋白在镍氢离子柱上纯化并成功复性。
本研究发现融合蛋白PTA-P、PTA-DOM1和PTA-DOM2主要有以下特性:1.可以凝集兔血,但是不能凝集人血而且他们的凝血活性受到甘露糖的抑制。这些性质和天南星科其他的凝集素是相似的。2.这三种半夏凝集素蛋白对细交链孢、离蠕孢、新月弯孢的生长具有明显的抑制,而且PTA-P的抑菌活性要高于其他两种蛋白。在被测试的三种真菌中,PTA-P、PTA-DOM1和PTA-DOM2均对细交链孢显示了最高的敏感性,对新月弯孢的敏感性最低。用50μg蛋白处理真菌24小时后,发现真菌菌丝尖端发生肿大而且多生分枝。3.三种半夏凝聚素蛋白在20℃和60℃之间保持完整活性,当温度达到100℃时只保留25%的活性。4.三种半夏凝集素蛋白在pH4到pH9之间都显示了活性,在pH7.8是活性最高。研究发现三种半夏凝集素蛋白还可以抑制细菌的生长,暗示利用分子生物学手段将PTA－P及两个亚基基因导入植物使之产生抗病性可能将是一个可行的方案。
Lectins are naturally occurring proteins and glycoproteins which bind selectively and non-covalently to carbohydrate residues.They are widely distributed in living organisms including animals,plants,microorganism.The main characteristic of this class of protein is their ability to interact specifically with carbohydrates and to combine with glyco-components of the cell surface.
On the basis of recent advances in biochemistry and molecular cloning and structural analysis,virtually all currently known plant lectins can be classified into seven families according to structurally and evolutionarily-related proteins: amaranthins,Cucurbitaceae phloem lectins,lectins with hevein domain(s), jacalinrelated lectins,legume lectins,monocot mannose-binding lectins and type-2 ribosome-inactivating proteins.Pinellia ternata is a traditional Chinese medicinal plant species,belonging to the family Araceae.Araceous lectins are a group of proteins possessing at least one non-catalytic domain that binds reversibly to specific monoor oligo-saccharide(glycoconjugate).They readily agglutinated rabbit erythrocytes but were unable to agglutinate human erythrocytes irrespective of the blood group.
The property of Pinellia ternate angustate agglutinin(PTA) and its domains was investigate in this study.The pta cDNA sequence encoding for mature PTA protein and two domains were cloned into PET-28a plasmid and the resulting in pET-28a-PTA、pET-28a-DOM1 and pET-28a-DOM2 containing N-terminal His-tagged fusion protein,repectiveLy.The expressions of them in E.coli BL21 were investigated.Purification of the PTA-P、PTA-DOM1 and PTA-DOM2 recombinant proteins were performed under native conditions by nickel ion metal-affinity chromatography.
The recombinant PTA-P、PTA-DOM1 and PTA-DOM2 exhibited several characteristics:1.they exhibited hemagglutinating activity towards rabbit but not human erythrocytes,and their hemagglutinating activity could be inhibited by mannose.These profiles were similar to other Araceae lectins.2.Both PTA-P and two domains exhibited potent antitungal activity toward Alternaria altanata,Bipolaris sorokiniana and Curvularia lunata.The antifungal activities of PTA-P were obviously stronger than that of PTA-DOM1 and PTA-DOM2.Among the tested fungi, Alternaria altanata appeared the most sensitive to PTA-P with an IC50 of 0.19μM, Alternaria altanata and Bipolaris sorokiniana more sensitive than Curvularia lunata to PTA、PTA-DOM1 and PTA-DOM2.The morphological toxicity of PTA-P、PTA-DOM1 and PTA-DOM1 to Curvularia lunata was revealed by an increase in mycelial apex offshoot and tumescence after treatment with 50ug proteins for 24 h.3. The results of thermal denaturation of fusion proteins showed that the antifungal activity of PTA-P、PTA-DOM1 and PTA-DOM1 were extremely stable between 20℃and 60℃.Even heating at 100℃for 5 min caused loss of only 25%of its original activities.4.they also exhibited activity under a broad pH range from 4.0 to 9.0 with highest activity at 7.8.We also found that the three proteins were active against Escherichia coli and Stapylococcus aureus.Sum up,The PTA-P,PTA-DOM1 and PTA-DOM1 may be a promising candidate for possible applications in the genetic engineering of disease-resistant crops.
引文
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