玉帘凝集素的分离纯化、部分性质研究及分子克隆
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摘要
本文对玉帘凝集素(Zephyranthes candida Herb Agglutinin，ZCA)的研究分为两个部分。第一部分为ZCA的分离纯化及部分性质研究；第二部分为ZCA的分子克隆及序列分析。
玉帘球茎经捣碎、生理盐水浸取、硫酸铵分级沉淀、DEAE-Sepharose离子交换层析、Sephacryl S-100分子筛层析可纯化到ZCA纯品，经SDS-PAGE检测为单一蛋白染色带。Sephacryl S-100分子筛凝胶过滤测得ZCA的表观分子量为48KD，SDS-PAGE显示其亚基分子量约为12.5KD，证明ZCA分子为由四个相同亚基组成的四聚体，用比色法测得每分子ZCA含有12.2个色氨酸残基，即每个亚基有3个，且色氨酸残基均位于分子表面或浅沟内。
ZCA具有很强的兔血凝集活性，在浓度为0.95μg／ml时仍能凝集兔红细胞，同时ZCA还具有凝集大肠杆菌和多种酵母细胞的作用。甘露聚糖和猪甲状腺球蛋白对ZCA的兔红细胞凝集活性有明显的抑制作用。研究了不同温度和pH对ZCA的荧光光谱和兔血凝集活性的影响，结果表明温度在80℃以上时分子构象发生了较大变化，但凝血活性仍保留50％的活性；在pH 4和pH 12时分子构象发生较大变化，但凝血活性只下降了27.3％，在pH 2时，凝血活性仍保留55％。所以ZCA具有很强的温度和pH耐受性。
荧光淬灭研究表明CsCl可以淬灭71％色氨酸的荧光，化学修饰实验表明N-溴代丁二酰亚胺(NBS)修饰色氨酸和2，3-丁二酮修饰精氨酸后，ZCA凝血活性明显下降，Ser／Thr被修饰后，凝血活性无明显变化。ZCA抗病毒实验结果表明，ZCA溶液在浓度为500μg／ml时无细胞毒性，浓度为5μg／ml以上时有
抗HSV一11活性，ICS。=5一1 op岁ml之间。
使用妞工49lA蛋白测序仪测定Z以的N一端和C一端部分序列显示，N-末端
前巧个氨基酸的序列为:D一S一I一L一Y一S{一E一T一L一S--A一司一S，C一末端7个氨基
酸残基的序列为:G一T一A礴--K一P.与石蒜科其它甘露糖结合凝集素序列进行比
较，发现有较高的同源性.
依据ZCA的N一端部分氨基酸设计简并引物，通过反转录、RT一CR与3’-
末端快速扩增法(3’一RACE)和5’一末端快速扩增法(5’一RACE)扩增得到
ZCA全长eDNA(Genbank注册号AF503622)，该。DNA全长为661bp，开放阅读
框从40一543bp，长504bp，编码168个氨基酸。起始密码子位于37一39bp处，
终止密码子位于544一546bp处。5’端非翻译区长为94饰，其后还有一段真核
生物mRNA所特有的poly(A)序列。根据对Z以的N一末端和C一末端部分氨基
酸序列的测定，发现Z以基因编码一前体蛋白:含有信号肤序列、成熟蛋白序
列和C一末端剪切序列。成熟蛋白由106个氨基酸残基组成，分子量为11甲7KD。
经在NCBI上的Blast进行比较发现，该成熟蛋白编码氨基酸序列与雪花莲凝
集素(GNA)同源性达到75.2%，与水仙凝集素同源性为72.4%;与石蒜凝集
素同源性为76.2%，与君子兰凝集素同源性为76.2%。在blocks数据库
(~blocks.伍crc.org)中检索zCA蛋白氨基酸序列的结构域，发现zCA氨
基酸序列中存在三个凝集素功能结构域区，并具有3个典型的甘露糖专一结合
位点盒(QDNY)。成熟蛋白质序列中，疏水氨基酸占38.7%，亲水氨基酸占43.4
%，碱性氨基酸占10.4%，酸性氨基酸占75%。Z以的信号肤包括24个氨基
酸，其中含一个9个氨基酸组成的疏水核心。由DNA PCR实验可知，ZCA
基因的编码区无内含子。此基因的克隆，为深入研究ZCA结构基因，剪切机
制，表达和调控机制以及ZCA结构和功能的关系莫定了重要的基础。
The study on Zephyranthes Candida Herb Agglutinin(ZCA) included two parts.The first part was the study on purification and characterization of ZCA, the second included the study on molecular cloning and sequence analysis of ZCA.
ZCA was isolated from bulbs of Zephyranthes Candida Herb by extraction,fraction with (NHOaSO-t, and followed by combination of ion-exchange chromatography on DEAE-Sepharose Fast Flow and gel filtration on Sephacryl S-100. The purified ZCA gave one band on SDS-acrylamide gel electrophoresis. The molecular weight was 48 KD determined by gel filtration. The subunit molecular weight was 12.5KD on SDS-PAGE. The results included that there were four same subunits in ZCA molecule.There were 12.2 Trp residues in a ZCA molecule , determinded by colorimetry and all the Trp residues are on the surface of ZCA.
ZCA could agglutinate rabbit erythrocytes at 0.95 g/mK E.coli cell at 1.95 g/ml and some sorts of Yeast cells . The result of carbohydrate inhibition assayed that Mannan and Thyroglobulin could inhibit the hemagglutination activity of ZCA. The study on the fluorescence spectra and the agglutinate activity of ZCA under different temperature and pH showed that ZCA was stable in various temperature and pH between pH 4-9.5 and below 80 , but the molecular structure and biological activity changed much under pH 2, pH 4, pH 12 and 80 , and the
activity of ZC A became weak.
The study on fluorescence quenching showed that 71% Trp could be quenched by CsCl. Chemical modification indicated that the Arginine and Tryptophan are essential for the hemagglutination activity of ZCA. The modification of Arginine and Tryptophan resulted the loss of rabbit hemaggulutinating activity. The modification of Ser/Thr didn't effect the hemagglutinating activity of ZCA. ZCA could inhibited the infection of the vero cells by Herpes simplex Virus type n(HSV-II) at IC50 values of 5~ 10 ug/ml and it showed no cytotoxic activity to vero cell proliferation at 500 g/ml.
The N-terminal amino acids were D-S-I-L-Y-S-G-E-T-L-S-A-G-Q-S; the C-terminal amino acids were G-T-A-W-K-A-P determined by amino acid sequence analysis. The sequence shared high homology with other Amaryllidaceae mannose-binding lectins.
Degenerate primers were designed in accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by RT-PCR, 3'-RACE and 5'-RACE, and sequenced (Genbank AF503622). The full-length cDNAhad 661 bp, and the sequence encoded an open reading frame of 168 amino acids..The start codon was at 37-39 bp and the stop codon was at 544-546 bp. The result showed that ZCA gene encoded a protein precursor with a signal peptide, mature protein and C-terminal cleavage amino acids sequence by the study of N-terminal and C-terminal partial amino acids sequence of ZCA. The mature protein included 106 amino acids residues and the molecular weight is 11.7KD . The mature protein sequence showed the identity to those of Galanthus nivalis agglutinin, Narcissus hybrid cultivar agglutinin , Lycoris radiate agglutinin, Clivia miniata agglutinin respectively are 75.2%, 72.4%, 76.2%, 76.2% , Blocks'analysis revealed that the deduced amino acid sequence of ZCA had three functional domains specific for lectin and three sugar-binding boxes(QDNY).In the mature protein,there were 38.7% hydrophobic amino acids, 43.4% hydrophilic amino acids, 10.4% basic amino acids and 7.5% acidic amino acids. The signal peptide of ZCA had 24 amino
acids,included a hydrophobia core of 9 ammo acids. There was no intron in the ZCA gene by the DNA PCR. The cloning of this gene established a important base to the study of ZCA gene structure, the cleavage mechanism, the mechanism of expression and regulation, the relationship of structure and function of ZCA
玉帘球茎经捣碎、生理盐水浸取、硫酸铵分级沉淀、DEAE-Sepharose离子交换层析、Sephacryl S-100分子筛层析可纯化到ZCA纯品，经SDS-PAGE检测为单一蛋白染色带。Sephacryl S-100分子筛凝胶过滤测得ZCA的表观分子量为48KD，SDS-PAGE显示其亚基分子量约为12.5KD，证明ZCA分子为由四个相同亚基组成的四聚体，用比色法测得每分子ZCA含有12.2个色氨酸残基，即每个亚基有3个，且色氨酸残基均位于分子表面或浅沟内。
ZCA具有很强的兔血凝集活性，在浓度为0.95μg／ml时仍能凝集兔红细胞，同时ZCA还具有凝集大肠杆菌和多种酵母细胞的作用。甘露聚糖和猪甲状腺球蛋白对ZCA的兔红细胞凝集活性有明显的抑制作用。研究了不同温度和pH对ZCA的荧光光谱和兔血凝集活性的影响，结果表明温度在80℃以上时分子构象发生了较大变化，但凝血活性仍保留50％的活性；在pH 4和pH 12时分子构象发生较大变化，但凝血活性只下降了27.3％，在pH 2时，凝血活性仍保留55％。所以ZCA具有很强的温度和pH耐受性。
荧光淬灭研究表明CsCl可以淬灭71％色氨酸的荧光，化学修饰实验表明N-溴代丁二酰亚胺(NBS)修饰色氨酸和2，3-丁二酮修饰精氨酸后，ZCA凝血活性明显下降，Ser／Thr被修饰后，凝血活性无明显变化。ZCA抗病毒实验结果表明，ZCA溶液在浓度为500μg／ml时无细胞毒性，浓度为5μg／ml以上时有
抗HSV一11活性，ICS。=5一1 op岁ml之间。
使用妞工49lA蛋白测序仪测定Z以的N一端和C一端部分序列显示，N-末端
前巧个氨基酸的序列为:D一S一I一L一Y一S{一E一T一L一S--A一司一S，C一末端7个氨基
酸残基的序列为:G一T一A礴--K一P.与石蒜科其它甘露糖结合凝集素序列进行比
较，发现有较高的同源性.
依据ZCA的N一端部分氨基酸设计简并引物，通过反转录、RT一CR与3’-
末端快速扩增法(3’一RACE)和5’一末端快速扩增法(5’一RACE)扩增得到
ZCA全长eDNA(Genbank注册号AF503622)，该。DNA全长为661bp，开放阅读
框从40一543bp，长504bp，编码168个氨基酸。起始密码子位于37一39bp处，
终止密码子位于544一546bp处。5’端非翻译区长为94饰，其后还有一段真核
生物mRNA所特有的poly(A)序列。根据对Z以的N一末端和C一末端部分氨基
酸序列的测定，发现Z以基因编码一前体蛋白:含有信号肤序列、成熟蛋白序
列和C一末端剪切序列。成熟蛋白由106个氨基酸残基组成，分子量为11甲7KD。
经在NCBI上的Blast进行比较发现，该成熟蛋白编码氨基酸序列与雪花莲凝
集素(GNA)同源性达到75.2%，与水仙凝集素同源性为72.4%;与石蒜凝集
素同源性为76.2%，与君子兰凝集素同源性为76.2%。在blocks数据库
(~blocks.伍crc.org)中检索zCA蛋白氨基酸序列的结构域，发现zCA氨
基酸序列中存在三个凝集素功能结构域区，并具有3个典型的甘露糖专一结合
位点盒(QDNY)。成熟蛋白质序列中，疏水氨基酸占38.7%，亲水氨基酸占43.4
%，碱性氨基酸占10.4%，酸性氨基酸占75%。Z以的信号肤包括24个氨基
酸，其中含一个9个氨基酸组成的疏水核心。由DNA PCR实验可知，ZCA
基因的编码区无内含子。此基因的克隆，为深入研究ZCA结构基因，剪切机
制，表达和调控机制以及ZCA结构和功能的关系莫定了重要的基础。
The study on Zephyranthes Candida Herb Agglutinin(ZCA) included two parts.The first part was the study on purification and characterization of ZCA, the second included the study on molecular cloning and sequence analysis of ZCA.
ZCA was isolated from bulbs of Zephyranthes Candida Herb by extraction,fraction with (NHOaSO-t, and followed by combination of ion-exchange chromatography on DEAE-Sepharose Fast Flow and gel filtration on Sephacryl S-100. The purified ZCA gave one band on SDS-acrylamide gel electrophoresis. The molecular weight was 48 KD determined by gel filtration. The subunit molecular weight was 12.5KD on SDS-PAGE. The results included that there were four same subunits in ZCA molecule.There were 12.2 Trp residues in a ZCA molecule , determinded by colorimetry and all the Trp residues are on the surface of ZCA.
ZCA could agglutinate rabbit erythrocytes at 0.95 g/mK E.coli cell at 1.95 g/ml and some sorts of Yeast cells . The result of carbohydrate inhibition assayed that Mannan and Thyroglobulin could inhibit the hemagglutination activity of ZCA. The study on the fluorescence spectra and the agglutinate activity of ZCA under different temperature and pH showed that ZCA was stable in various temperature and pH between pH 4-9.5 and below 80 , but the molecular structure and biological activity changed much under pH 2, pH 4, pH 12 and 80 , and the
activity of ZC A became weak.
The study on fluorescence quenching showed that 71% Trp could be quenched by CsCl. Chemical modification indicated that the Arginine and Tryptophan are essential for the hemagglutination activity of ZCA. The modification of Arginine and Tryptophan resulted the loss of rabbit hemaggulutinating activity. The modification of Ser/Thr didn't effect the hemagglutinating activity of ZCA. ZCA could inhibited the infection of the vero cells by Herpes simplex Virus type n(HSV-II) at IC50 values of 5~ 10 ug/ml and it showed no cytotoxic activity to vero cell proliferation at 500 g/ml.
The N-terminal amino acids were D-S-I-L-Y-S-G-E-T-L-S-A-G-Q-S; the C-terminal amino acids were G-T-A-W-K-A-P determined by amino acid sequence analysis. The sequence shared high homology with other Amaryllidaceae mannose-binding lectins.
Degenerate primers were designed in accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by RT-PCR, 3'-RACE and 5'-RACE, and sequenced (Genbank AF503622). The full-length cDNAhad 661 bp, and the sequence encoded an open reading frame of 168 amino acids..The start codon was at 37-39 bp and the stop codon was at 544-546 bp. The result showed that ZCA gene encoded a protein precursor with a signal peptide, mature protein and C-terminal cleavage amino acids sequence by the study of N-terminal and C-terminal partial amino acids sequence of ZCA. The mature protein included 106 amino acids residues and the molecular weight is 11.7KD . The mature protein sequence showed the identity to those of Galanthus nivalis agglutinin, Narcissus hybrid cultivar agglutinin , Lycoris radiate agglutinin, Clivia miniata agglutinin respectively are 75.2%, 72.4%, 76.2%, 76.2% , Blocks'analysis revealed that the deduced amino acid sequence of ZCA had three functional domains specific for lectin and three sugar-binding boxes(QDNY).In the mature protein,there were 38.7% hydrophobic amino acids, 43.4% hydrophilic amino acids, 10.4% basic amino acids and 7.5% acidic amino acids. The signal peptide of ZCA had 24 amino
acids,included a hydrophobia core of 9 ammo acids. There was no intron in the ZCA gene by the DNA PCR. The cloning of this gene established a important base to the study of ZCA gene structure, the cleavage mechanism, the mechanism of expression and regulation, the relationship of structure and function of ZCA
引文
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