Abstract：
BACKGROUND: Artemisia species are widely spread in north hemisphere. Artemisia sieversiana pollen is one of the common pollen allergens in the north of China. At present, seven allergens were identified and had been listed officially from A. sieversiana pollen, but the remaining allergens are still insufficiently studied, which need to be found.
METHODS: Pectate lyase was purified from the extracts of A. sieversiana pollen by anion exchange, size exclusion, and HPLC-hydrophobic interaction chromatography. The gene of A. sieversiana pectate lyase (Art si pectate lyase) was cloned and expressed in Escherichia coli. The enzyme activity and circular dichroism (CD) spectrum of natural and recombinant proteins were analyzed. The allergenicity of Art si pectate lyase was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test. The allergen\'s physicochemical properties, three-dimensional structure, sequence profiles with homologous allergens and phylogenetic tree were analyzed by in silico methods.
RESULTS: Natural Art si pectate lyase (nArt si pectate lyase) was purified from A. sieversiana pollen extracts by three chromatographic strategies. The cDNA sequence of Art si pectate lyase had a 1191-bp open reading frame encoding 396 amino acids. Both natural and recombinant pectate lyase (rArt si pectate lyase) exhibited similar CD spectrum, and nArt si pectate lyase had higher enzymatic activity. Moreover, the specific immunoglobulin E (IgE) binding rate against nArt si pectate lyase and rArt si pectate lyase was determined as 40% (6/15) in patients\' serum with Artemisia species pollen allergy by ELISA. The nArt si pectate lyase and rArt si pectate lyase could inhibit 76.11% and 47.26% of IgE binding activities to the pollen extracts, respectively. Art si pectate lyase was also confirmed to activate patients\' basophils. Its structure contains a predominant motif of classic parallel helical core, consisting of three parallel β-sheets, and two highly conserved features (vWiDH, RxPxxR) which may contribute to pectate lyase activity. Moreover, Art si pectate lyase shared the highest sequence identity of 73.0% with Art v 6 among currently recognized pectate lyase allergen, both were clustered into the same branch in the phylogenetic tree.
CONCLUSIONS: In this study, pectate lyase was identified and comprehensively characterized as a novel allergen in A. sieversiana pollen. The findings enriched the allergen information for this pollen and promoted the development of component-resolved diagnosis and molecular therapy of A. sieversiana pollen allergy.
METHODS: Pectate lyase was purified from the extracts of A. sieversiana pollen by anion exchange, size exclusion, and HPLC-hydrophobic interaction chromatography. The gene of A. sieversiana pectate lyase (Art si pectate lyase) was cloned and expressed in Escherichia coli. The enzyme activity and circular dichroism (CD) spectrum of natural and recombinant proteins were analyzed. The allergenicity of Art si pectate lyase was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test. The allergen\'s physicochemical properties, three-dimensional structure, sequence profiles with homologous allergens and phylogenetic tree were analyzed by in silico methods.
RESULTS: Natural Art si pectate lyase (nArt si pectate lyase) was purified from A. sieversiana pollen extracts by three chromatographic strategies. The cDNA sequence of Art si pectate lyase had a 1191-bp open reading frame encoding 396 amino acids. Both natural and recombinant pectate lyase (rArt si pectate lyase) exhibited similar CD spectrum, and nArt si pectate lyase had higher enzymatic activity. Moreover, the specific immunoglobulin E (IgE) binding rate against nArt si pectate lyase and rArt si pectate lyase was determined as 40% (6/15) in patients\' serum with Artemisia species pollen allergy by ELISA. The nArt si pectate lyase and rArt si pectate lyase could inhibit 76.11% and 47.26% of IgE binding activities to the pollen extracts, respectively. Art si pectate lyase was also confirmed to activate patients\' basophils. Its structure contains a predominant motif of classic parallel helical core, consisting of three parallel β-sheets, and two highly conserved features (vWiDH, RxPxxR) which may contribute to pectate lyase activity. Moreover, Art si pectate lyase shared the highest sequence identity of 73.0% with Art v 6 among currently recognized pectate lyase allergen, both were clustered into the same branch in the phylogenetic tree.
CONCLUSIONS: In this study, pectate lyase was identified and comprehensively characterized as a novel allergen in A. sieversiana pollen. The findings enriched the allergen information for this pollen and promoted the development of component-resolved diagnosis and molecular therapy of A. sieversiana pollen allergy.
摘要：
背景：蒿属广泛分布于北半球。黄蒿花粉是我国北方常见的花粉过敏原之一。目前,已经确定了7种过敏原，并已从A.sieversiana花粉中正式列出，但是其余的过敏原仍然没有得到充分的研究，需要找到。
方法：通过阴离子交换从A.sieversiana花粉提取物中纯化果胶酸裂解酶，尺寸排除，和HPLC-疏水相互作用色谱。克隆了A.sieversiana果胶酸裂解酶（Artsi果胶酸裂解酶）的基因，并在大肠杆菌中表达。分析了天然和重组蛋白的酶活性和圆二色性（CD）谱。通过酶联免疫吸附试验（ELISA）表征了Artsi果胶酸裂解酶的致敏性，蛋白质印迹,抑制ELISA,和嗜碱性粒细胞激活试验.过敏原的理化性质，三维结构，通过计算机模拟方法分析了具有同源过敏原和系统发育树的序列谱。
结果：通过三种色谱策略从A.sieversiana花粉提取物中纯化了天然的果胶酸裂解酶（nArtsi果胶酸裂解酶）。Artsi果胶酸裂解酶的cDNA序列具有编码396个氨基酸的1191-bp开放阅读框。天然和重组果胶酸裂解酶（rArtsi果胶酸裂解酶）均表现出相似的CD谱，nArtsi果胶酸裂解酶具有较高的酶活性。此外，通过ELISA测定，在患有蒿属花粉过敏的患者血清中，针对nArtsi果胶酸裂解酶和rArtsi果胶酸裂解酶的特异性免疫球蛋白E（IgE）结合率为40％（6/15）。nArtsi果胶酸裂解酶和rArtsi果胶酸裂解酶可以抑制花粉提取物中IgE结合活性的76.11%和47.26%，分别。果胶酸裂解酶也被证实激活患者的嗜碱性粒细胞。它的结构包含经典平行螺旋芯的主要基序，由三个平行的β-折叠组成，和两个高度保守的特征(vWiDH，RxPxxR）可能有助于果胶酸裂解酶活性。此外，在目前公认的果胶酸裂解酶过敏原中，Artsi果胶酸裂解酶与Artv6共享73.0％的最高序列同一性，两者都聚集在系统发育树中的同一分支中。
结论：在这项研究中，果胶酸裂解酶被鉴定并全面表征为A.sieversiana花粉中的新型过敏原。该发现丰富了该花粉的过敏原信息，并促进了Sieversiana花粉过敏的成分分辨诊断和分子治疗的发展。
方法：通过阴离子交换从A.sieversiana花粉提取物中纯化果胶酸裂解酶，尺寸排除，和HPLC-疏水相互作用色谱。克隆了A.sieversiana果胶酸裂解酶（Artsi果胶酸裂解酶）的基因，并在大肠杆菌中表达。分析了天然和重组蛋白的酶活性和圆二色性（CD）谱。通过酶联免疫吸附试验（ELISA）表征了Artsi果胶酸裂解酶的致敏性，蛋白质印迹,抑制ELISA,和嗜碱性粒细胞激活试验.过敏原的理化性质，三维结构，通过计算机模拟方法分析了具有同源过敏原和系统发育树的序列谱。
结果：通过三种色谱策略从A.sieversiana花粉提取物中纯化了天然的果胶酸裂解酶（nArtsi果胶酸裂解酶）。Artsi果胶酸裂解酶的cDNA序列具有编码396个氨基酸的1191-bp开放阅读框。天然和重组果胶酸裂解酶（rArtsi果胶酸裂解酶）均表现出相似的CD谱，nArtsi果胶酸裂解酶具有较高的酶活性。此外，通过ELISA测定，在患有蒿属花粉过敏的患者血清中，针对nArtsi果胶酸裂解酶和rArtsi果胶酸裂解酶的特异性免疫球蛋白E（IgE）结合率为40％（6/15）。nArtsi果胶酸裂解酶和rArtsi果胶酸裂解酶可以抑制花粉提取物中IgE结合活性的76.11%和47.26%，分别。果胶酸裂解酶也被证实激活患者的嗜碱性粒细胞。它的结构包含经典平行螺旋芯的主要基序，由三个平行的β-折叠组成，和两个高度保守的特征(vWiDH，RxPxxR）可能有助于果胶酸裂解酶活性。此外，在目前公认的果胶酸裂解酶过敏原中，Artsi果胶酸裂解酶与Artv6共享73.0％的最高序列同一性，两者都聚集在系统发育树中的同一分支中。
结论：在这项研究中，果胶酸裂解酶被鉴定并全面表征为A.sieversiana花粉中的新型过敏原。该发现丰富了该花粉的过敏原信息，并促进了Sieversiana花粉过敏的成分分辨诊断和分子治疗的发展。
