黑斑蛙皮肤生物活性肽的分离纯化、结构鉴定及生物活性检测
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摘要
两栖动物是最原始的陆生脊椎动物,既有适应陆地生活的新的性状,又有从鱼类祖先继承下来的适应水生生活的性状。两栖类动物皮肤裸露,为了适应复杂多样的生态环境并维持自身的生存,皮肤起到重要作用。两栖类皮肤可以分泌大量功能复杂、种类繁多的生物活性物质,对抵御有害环境因子侵袭有着重要作用。蛙类皮肤活性肽资源丰富包括抗菌肽、舒缓激肽样多肽、血管紧张素因子、多肽生长因子、吗啡因子、保湿因子等。生物活性多肽具有分子和功能的多样性。两栖类皮肤资源丰富,可用于生物活性多肽的发现、纯化和功能研究。黑斑蛙(Rana nigromaculata)属于蛙科、蛙属,广泛分布在我国各地,资源丰富。
本论文选取辽宁省大连市金州登沙河镇的黑斑蛙为实验材料,运用反相高效液相色谱技术对黑斑蛙皮肤分泌物中生物活性多肽进行提取和纯化,获得6种皮肤生物活性肽。利用MS和Q-TOF MS/MS对6种多肽的分子量和氨基酸序列进行了测定。首先利用电刺激诱导方法取得黑斑蛙的皮肤分泌液。采用VydacTP510反相高效液相色谱,对皮肤分泌液采用线性梯度洗脱,分离纯化得到6个多肽。6条多肽分子量从1060.2到3245.9道尔顿,由9个到32个氨基酸组成。其一级结构分别为RPPGFSPFR、RPPGFSDSSPLAPGT、LKNVGKEVGFDVENELRLAGHARPLGK、KSPGVGALAGKK PHGLLLSGLKNVGGAEAGFD、LGLGKVLGVGQRALAHSPPK、和LVPLVSYHLPVL。通过比对NCBI蛋白质数据库,由9个氨基酸组成的多肽RPPGFSPFR是典型缓激肽(Bradykinin),命名为Bradykinin-RN;RPPGFSDSSPLAPGT具有缓激肽的结构域保守序列RPPGFS-,属于缓激肽相关肽BRPs(Bradykinin-related peptides),命名为Ranakinin-RN1。LVPLVSYHLPVL虽然不含有缓激肽的保守序列,但具有较高的缓激肽活性,所以命名为Ranakinin-RN2,而其余多肽同已知其它两栖类皮肤多肽相似度很低,属于新的多肽,分别命名为:Nigromin-1,Nigromin-2和Nigromin-3。抗菌活性测定结果显示,这3个新肽对2种细菌均没有抑制作用。对3个缓激肽和缓激肽相关肽进行豚鼠离体回肠实验测定对肠肌收缩性的影响。实验结果显示Bradykinin-RN和Ranakinin-RN1豚鼠回肠的收缩强度呈现一个正的线性关系,Bradykinin-RN的缓激肽活性比缓激肽Ranakinin-RN1强。Ranakinin-RN2随着浓度提高,其缓激肽活性达到最高后活性受到抑制。
Amphibians is the most primitive terrestrial vertebrates adapt to both terrestrial and aquatic life. Amphibians skin is uncovered and without hair, feathers and shells. Amphibians skin play an important role in the complex and diverse environment to maintain its own living. Amphibians skin contains biological vivid substances with various comploicated biofunctions, and these substances play an important role in resisting the invasion of harmful environmental factors. Meanwhile, the skin of different Amphibians species consists of different bioactive substances. Amphibian’s skin is rich in natural resources and used in new bioactive peptides’discovery, refiner and function research. Rana nigromaculata of China is one of animal resource, which has medical value in northeast regionof china. Researches have shown that Rana nigromaculata skin contains various active peptides, such as bradykinin-like peptide, bombesin peptide, growth factor, morphine and moisturizing factors.
In this paper, Rana nigromaculata growth in Jinzhou Dengshahe of Dalian of Liaoning was choosed as the experimental material. The isolation, purification and structural characterization of bioactive peptides from the skin of Rana nigromaculata were carried out by gel chromatography and RP-HPLC. The optimized conditions of purification were screened, and seven of skin bioactive peptides were obtained. MS and Q-TOF MS/MS were used to determinate their molecular weight and amino acid sequences. Rana nigromaculata skin secretion was obtained by electrical stimulation. The secretion was purified using VydacTP510 RP-HPLC into 6 single peptides.The results of analysis of amino acid sequences showed that three of these peptides were bradykinin or bradykinin-related peptides, and the other three were antimicrobial peptides. Their molecular weights were from 1060.2 to 3285.96 Dalton, and composed of 9 to 32 amino acids. Their primary structure was RPPGFSPFR,,RPPGFSDSSPLAPGT,LKNVGKEVGFDVENELRLAGHARPLGK,KSPGV GALAGKKPHGLLLSGLKNVGGAEAGFD,LGLGKVLGVGQRALAHSPPK and LVPLV SYHLPVL. After BLAST searched in NCBI Protein Databases, we found that peptides RPPGFSPFR was typical Bradykinin composed of 9 amino acids and RPPGFSDSSPLAPGT had the motif of Bradykinin (RPPGFS-) and belonged to bradykinin-related peptides,named it Ranakinin-RN1.Although the peptide LVPLVSYHLPVL does not contain the conserved domain of bradykinin, we still named it as Ranakinin-RN2 due to its higher homology bradykinin activity. Meanwhile, the rest of peptides shared a lower homoloy with some known amphibian skin peptides. Therefore, they are the new peptides, named them as nigromin-1,nigromin-2, nigromin-3, respectively.
The bradykinin activity of bradykinin and bradykinin-related peptides was tested by assaying the contractile activity on isolated guinea pig ileum. The results showed Bradykinin-RN and Ranakinin-RN1 contracted guinea pig ileum in a positive linear relationship, bradykinin activity of Bradykinin-RN was more potent than Ranakinin-RN1. bradykinin activity of Ranakinin-RN2 was inhibited after reaching its maximum, showing that the peptide might be the bradykinin antagonist peptide.
本论文选取辽宁省大连市金州登沙河镇的黑斑蛙为实验材料,运用反相高效液相色谱技术对黑斑蛙皮肤分泌物中生物活性多肽进行提取和纯化,获得6种皮肤生物活性肽。利用MS和Q-TOF MS/MS对6种多肽的分子量和氨基酸序列进行了测定。首先利用电刺激诱导方法取得黑斑蛙的皮肤分泌液。采用VydacTP510反相高效液相色谱,对皮肤分泌液采用线性梯度洗脱,分离纯化得到6个多肽。6条多肽分子量从1060.2到3245.9道尔顿,由9个到32个氨基酸组成。其一级结构分别为RPPGFSPFR、RPPGFSDSSPLAPGT、LKNVGKEVGFDVENELRLAGHARPLGK、KSPGVGALAGKK PHGLLLSGLKNVGGAEAGFD、LGLGKVLGVGQRALAHSPPK、和LVPLVSYHLPVL。通过比对NCBI蛋白质数据库,由9个氨基酸组成的多肽RPPGFSPFR是典型缓激肽(Bradykinin),命名为Bradykinin-RN;RPPGFSDSSPLAPGT具有缓激肽的结构域保守序列RPPGFS-,属于缓激肽相关肽BRPs(Bradykinin-related peptides),命名为Ranakinin-RN1。LVPLVSYHLPVL虽然不含有缓激肽的保守序列,但具有较高的缓激肽活性,所以命名为Ranakinin-RN2,而其余多肽同已知其它两栖类皮肤多肽相似度很低,属于新的多肽,分别命名为:Nigromin-1,Nigromin-2和Nigromin-3。抗菌活性测定结果显示,这3个新肽对2种细菌均没有抑制作用。对3个缓激肽和缓激肽相关肽进行豚鼠离体回肠实验测定对肠肌收缩性的影响。实验结果显示Bradykinin-RN和Ranakinin-RN1豚鼠回肠的收缩强度呈现一个正的线性关系,Bradykinin-RN的缓激肽活性比缓激肽Ranakinin-RN1强。Ranakinin-RN2随着浓度提高,其缓激肽活性达到最高后活性受到抑制。
Amphibians is the most primitive terrestrial vertebrates adapt to both terrestrial and aquatic life. Amphibians skin is uncovered and without hair, feathers and shells. Amphibians skin play an important role in the complex and diverse environment to maintain its own living. Amphibians skin contains biological vivid substances with various comploicated biofunctions, and these substances play an important role in resisting the invasion of harmful environmental factors. Meanwhile, the skin of different Amphibians species consists of different bioactive substances. Amphibian’s skin is rich in natural resources and used in new bioactive peptides’discovery, refiner and function research. Rana nigromaculata of China is one of animal resource, which has medical value in northeast regionof china. Researches have shown that Rana nigromaculata skin contains various active peptides, such as bradykinin-like peptide, bombesin peptide, growth factor, morphine and moisturizing factors.
In this paper, Rana nigromaculata growth in Jinzhou Dengshahe of Dalian of Liaoning was choosed as the experimental material. The isolation, purification and structural characterization of bioactive peptides from the skin of Rana nigromaculata were carried out by gel chromatography and RP-HPLC. The optimized conditions of purification were screened, and seven of skin bioactive peptides were obtained. MS and Q-TOF MS/MS were used to determinate their molecular weight and amino acid sequences. Rana nigromaculata skin secretion was obtained by electrical stimulation. The secretion was purified using VydacTP510 RP-HPLC into 6 single peptides.The results of analysis of amino acid sequences showed that three of these peptides were bradykinin or bradykinin-related peptides, and the other three were antimicrobial peptides. Their molecular weights were from 1060.2 to 3285.96 Dalton, and composed of 9 to 32 amino acids. Their primary structure was RPPGFSPFR,,RPPGFSDSSPLAPGT,LKNVGKEVGFDVENELRLAGHARPLGK,KSPGV GALAGKKPHGLLLSGLKNVGGAEAGFD,LGLGKVLGVGQRALAHSPPK and LVPLV SYHLPVL. After BLAST searched in NCBI Protein Databases, we found that peptides RPPGFSPFR was typical Bradykinin composed of 9 amino acids and RPPGFSDSSPLAPGT had the motif of Bradykinin (RPPGFS-) and belonged to bradykinin-related peptides,named it Ranakinin-RN1.Although the peptide LVPLVSYHLPVL does not contain the conserved domain of bradykinin, we still named it as Ranakinin-RN2 due to its higher homology bradykinin activity. Meanwhile, the rest of peptides shared a lower homoloy with some known amphibian skin peptides. Therefore, they are the new peptides, named them as nigromin-1,nigromin-2, nigromin-3, respectively.
The bradykinin activity of bradykinin and bradykinin-related peptides was tested by assaying the contractile activity on isolated guinea pig ileum. The results showed Bradykinin-RN and Ranakinin-RN1 contracted guinea pig ileum in a positive linear relationship, bradykinin activity of Bradykinin-RN was more potent than Ranakinin-RN1. bradykinin activity of Ranakinin-RN2 was inhibited after reaching its maximum, showing that the peptide might be the bradykinin antagonist peptide.
引文
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[3]徐强,华跃进,徐步进等.蛙类皮肤分泌物中的抗菌肽和抗癌肽.动物学杂志,2002,37(2):73-76.
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