牛蛙（Rana catesbeiana）皮肤活性肽的分子克隆及其生物学活性研究
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摘要
本研究以牛蛙(Rana catesbeiana)皮肤中提取的mRNA为材料,构建牛蛙皮肤cDNA文库。根据GenBank已报道蛙属抗菌肽基因信号肽保守区域设计简并引物,利用PCR方法对文库进行筛选。结果得到48个完整的cDNA序列,分别编码24种抗菌肽前体,属于9个不同的抗菌肽家族。其中18个分别属于已知的Ranatuerin-1(6个)、Ranatuerin-2(3个)、Temporin(3个)、Palustrin(1个)、Ranatuerin-5(3个)、Ranatuerin-7(1个)、Ranatuerin-8(1个)家族,6个与已发现的抗菌肽同源性较低,为新发现的抗菌肽序列,根据序列特征分析结果将其归为以下3个家族: Catesbeianin-1 ( Catesbeianin-1 ); Ranacyclin(Ranacyclin-Ca、Ranacyclin-Cb与Ranacyclin-Cc);Temporin(Temporin-La、Temporin-Lb)。
对新发现的抗菌肽进行化学合成,通过生物学活性比较研究,得到了抗菌肽、胰蛋白酶抑制剂和凝集素三种生物活性肽。其中包括两个同时具有抗菌活性与胰蛋白酶抑制剂活性的多肽Ranacyclin-Ca与Ranacyclin-Cb,测定其对胰蛋白酶的抑制常数分别为7.02×10~7M与5.6×10~7M,为不可逆抑制并具有热稳定性。凝集素活性测定筛选到分子量为1471.8Da的牛蛙凝集素Temporin-1Ca,并利用激光共聚焦显微镜观察了凝集素Temporin-1Ca对金黄色葡萄球菌、猪链球菌2型、胸膜肺炎放线杆菌、仔猪副伤寒沙门菌的凝集活性。Temporin-Lb与Catesbeianin-1具有很强的DPPH自由基清除能力;Palustrin-Ca能够促进NO释放,当浓度为10μg/mL时,可以促进57.45%的NO释放。抗肿瘤细胞活性测定结果,Temporin-La对胃癌细胞株SMMC7721的IC_(50)为1.384μg/mL,而Palustrin-Ca对肝癌细胞株SGC7901的IC_(50)仅为0.951μg/ mL。抗SIV测定结果表明,在CEMX174细胞最大安全浓度下,Palustrin-Ca和Catesbeianin-1对SIV的抑制率较高,可达88.79%与84.30%。
通过随机筛选牛蛙皮肤cDNA文库,从牛蛙皮肤中得到牛蛙缓激肽Kininogen-1Ca的前体及cDNA序列,编码RR9(RPPGCNPFR)和RL16(RPPGCNPFRIAPASYL)的牛蛙缓激肽。结果显示具有明显的促进大鼠回肠肌收缩的作用。这是首次从牛蛙中得到的缓激肽类分子及其cDNA序列。
应用透射电镜对筛选到的4种牛蛙抗菌肽的抗菌机制进行了初步研究,并观察了牛蛙抗菌肽对金葡菌感染小鼠创面的治疗效果,结果表明合成的牛蛙抗菌肽Palustrin-Ca和Temporin-La对小鼠金葡萄感染创面有明显的抗感染作用并能促进创面的愈合。本研究为牛蛙皮肤抗菌活性肽的多样性提供了依据,同时也为研究多样性的两栖动物皮肤活性肽,发现新型抗菌、抗癌药物前体物质,设计新型活性肽建立了研究方法和参考。
A variety of bio-active substances from amphibian skin that play an important role in defense against invading factors have attracted significant attention of the researchers all over the world. So far, many kinds of proteins and polypeptides have been isolated and characterized from amphibian. Rana catesbeiana originally lived in Rocky Mountain north America, which were introduced into China in 1959, and have raised at a large amount in middle and south of China now. The antibacterial peptides from the skin of Rana catesbeiana play an essential role in amphibian innate immune system and reportedly have stronger killing effect than any other common drugs on, pseudomonas aeruginosa that could cause blind on man, but without problems of drug resistance.
To analyze the diversity and structures of the skin antibacterial peptides of Rana catesbeiana, Rana catesbeiana skin cDNA library was constructed with about 1×106 independent clones. According to GenBank, degenerate primers were designed for antibacterial peptides sequence of Rana catesbeiana based on the conservative region of signal peptide of Rana antibacterial peptides. Genes of antibacterial peptide were amplified from the library by PCR methods. One hundred of cDNA clones were sequenced, and 48 full-length cDNA sequences encoding 24 kinds of antimicrobial peptides precursor were obtained, among which 18 antimicrobial peptides precursor were subordinated by nine known antimicrobial families previously identified in the skins of Ranid frog s:Ranatuerin-1(6),Ranatuerin-2(3), temporin(3),Palustrin(1),Ranatuerin-5 (3),Ranatuerin-7(1),Ranatuerin-8(1)and other six anti-microbial peptides precursor had been newly found antimicrobial peptides which had much lower homology than others known,and were named and divided into three families: Catesbeianin-1(Catesbeianin-1), Ranacyclin(Ranacyclin-Ca,Cb and Cc),temporin (Temporin-La and Lb).The signal and propiece domain(SPD)of the peptides were extremely conserve while mature peptide domain had much more variation, that they were perhaps originated from a common ancestor. It was also supposed that formation of diversity of antimicrobial peptides have relationship with construction of microorganism in bullfrog’s living environment. Transition and transversion between every another nucleotide sequence of nine antimicrobial peptide families were conducted, the results suggested that there were difference substitution patterns in the two domains, perhaps because they suffered from different selective pressure in the evolution.
Seven newly found polypeptides named Temporin-La,Temporin-Lb,Catesbeian in-1,Ranacyclin-Ca, Ranacyclin-Cb,Palustrin-Ca,Temporin-1Ca were synthesized by chemosynthesis methods, Biological activities including antibacterial, hemolytic, hemagglutination, trypsin activity/trypsin-inhabiting,antioxidation, NO release, antitumor, antivirus were assayed and the results were as follows:
(1) For studying the antibacterial activities, Minimum Inhibitory Concentration (MIC) methods were determined, and the results showed that antimicrobial peptides of Temporin family had stronger inhibition activities on staphylococcus aureus and streptococcus suis. The MIC of staphylococcus aureus by Temporin-La was 2.5μg /mL and streptococcus suis by Temporin-Lb was 7.8μg/mL, Palustrin-Ca was 7.8μg/mL.
(2) Detection of hemolytic indicated that hemolytic rates of Catesbeianin-1, Temporin-1Ca, Palustrin-Ca for rabbit red blood cells were 0.8%, 0.30%, 0.25% respectively. Temporin-Lb have strong hemolytic activity with a hemolytic rate amounted to 31.56%, while that of Temporin-La was 1.75%. The C-end of the two peptides were both animated, variation of only two amino acids, Leu at 2, 9 site in Temporin-Lb was substituted with Phe at 2, 9 site. Although both of them were hydrophobic amino acids, their vicariousness may cause different hemolytic activity between the two peptides. Perhaps the differences of amino acids lead to differences of hemolytic activity, which would provide reference for molecular designing of antibacterial peptide.
(3)Detection of activities of trypsin and trypsin inhabitation showed that both Ranacyclin-Ca and Ranacyclin-Cb polypeptides had activities of s trypsin inhabitation. Compared to known trypsin inhibitor, there were two amino acids difference in the motifs of active region. Both of the Ranacyclin-Ca and Ranacyclin-Cb were thermal stability and effective irreversible trypsin inhabitation activity with a Ki value of 7.02×10~(-7)M and 5.6×10~(-7)M respectively.
(4) Erythrocyte agglutination activity detection showed that a lectin of Rana catesbeiana (Temporin-1Ca) with the molecular weight of 1471.8Da, which is smaller than any other known lectin from amphibian, has very low homology with the other lectin. Temporin-1Ca had very high rabbit erythrocyte agglutination activity with the minimum concentration 12.5μg/mL. It was also indicated that Temporin-1Ca had significant bacterial agglutination activity by laser scanning confocal microscope for Staphyloccocus aureus, streptococcus suis, Salmonella, Actinobacillus pleuropneu moniae.
(5) Temporin-Lb and Catesbeianin-1 had stronger elimination ability of DPPH with the rate of 76.50% and 69.47% respectively. Moreover, Palustrin-Ca was able to promote NO releasing. With a concentration of 10μg/mL, Palustrin-Ca can promote 57.45% of the NO release.
(6) Temporin-Lb and Palustrin-Ca had stronger anticancer activities, the IC_(50) of Temporin-Lb to gastric tumor cell SMMC7721 strain was 1.384μg/ mL, as well as the IC_(50) of Palustrin-Ca to the liver tumor cell SGC 7901 strain was 0.95μg/ mL.
(7)Antiviral activity detection showed that At its safe concentration,The inhibition rates of Palustrin-Ca, Catesbeianin-1 and Temporin-La to the SIV were 88.79%, 84.30% and 80.76%,respectively.
By random screening of cDNA library of bullfrog skin, a cDNA sequence of a kind of bradykinin-precurosor of Kininogen-1Ca from Rana catesbeiana were obtained, which had 77% identity with kininogen-1 precursor from Rana pipiens. The bradykinin which encoding RL16 ( RPPGCNPFRIAPASYL ) and RR9(RPPGCNPFR)was detected using biologic function radiomete on the influence of contraction ability of rat ileum muscle, bradykinin could contraction rat ileum muscle significantly. This is the first report of bradykinin in Rana catesbeiana and its cDNA sequence.
Summarizing above results, antimicrobial peptides, trypsin inhibitor, lectin and bradykinin were obtained from the skin of bull frog by random screening of cDNA library of bullfrog skin.
The effects of antibacterial peptide Temporin-La, Temporin-Lb, Palustrin-Ca, Catesbeianin-1 were observed that the antibacterial peptides had effect on microstructure of Staphylococcus aureus and streptococcus suis serotype 2 using transmission electron microscope.Temporin-La and Palustrin-Ca could caused the plasmolysis of Staphylococcus aureus ,while Temporin-Lb and Catesbeianin-1 could cause the bacteria formed a blurring fibrosis of bacterial cell wall between cell wall and cytomembrane. All the four peptides could cause cell wall clearage of streptococcus suis. There were various kinds of different mechanism of antibacterial peptide should be researched.
Temporin-La and Palustrin-Ca with high bacteriostasis activity were studied in treating infected wound in mice skin with Staphylococcus aureus, the number of bacteria in wound, the number of leucocytes, tissue slice, as well as the expression level of vascular endothelial growth factor (VEGF) were determined. Consequently, the results showed that the synthetical antibacterial peptide had significant effects of anti-inflammatory and the healing effect of Palustrin-Ca was better than that of Temporin-La as well as antibiotics treatment group.
The screened peptides studied in this study might become precursors for new drugs developing and provide potential new templates to further explore. In addition, this study provided a reference for the studying of the diversity of bioactive peptides of amphibians, new antibiotic or anticancer drugs developing and new type bioactive peptide designing.
对新发现的抗菌肽进行化学合成,通过生物学活性比较研究,得到了抗菌肽、胰蛋白酶抑制剂和凝集素三种生物活性肽。其中包括两个同时具有抗菌活性与胰蛋白酶抑制剂活性的多肽Ranacyclin-Ca与Ranacyclin-Cb,测定其对胰蛋白酶的抑制常数分别为7.02×10~7M与5.6×10~7M,为不可逆抑制并具有热稳定性。凝集素活性测定筛选到分子量为1471.8Da的牛蛙凝集素Temporin-1Ca,并利用激光共聚焦显微镜观察了凝集素Temporin-1Ca对金黄色葡萄球菌、猪链球菌2型、胸膜肺炎放线杆菌、仔猪副伤寒沙门菌的凝集活性。Temporin-Lb与Catesbeianin-1具有很强的DPPH自由基清除能力;Palustrin-Ca能够促进NO释放,当浓度为10μg/mL时,可以促进57.45%的NO释放。抗肿瘤细胞活性测定结果,Temporin-La对胃癌细胞株SMMC7721的IC_(50)为1.384μg/mL,而Palustrin-Ca对肝癌细胞株SGC7901的IC_(50)仅为0.951μg/ mL。抗SIV测定结果表明,在CEMX174细胞最大安全浓度下,Palustrin-Ca和Catesbeianin-1对SIV的抑制率较高,可达88.79%与84.30%。
通过随机筛选牛蛙皮肤cDNA文库,从牛蛙皮肤中得到牛蛙缓激肽Kininogen-1Ca的前体及cDNA序列,编码RR9(RPPGCNPFR)和RL16(RPPGCNPFRIAPASYL)的牛蛙缓激肽。结果显示具有明显的促进大鼠回肠肌收缩的作用。这是首次从牛蛙中得到的缓激肽类分子及其cDNA序列。
应用透射电镜对筛选到的4种牛蛙抗菌肽的抗菌机制进行了初步研究,并观察了牛蛙抗菌肽对金葡菌感染小鼠创面的治疗效果,结果表明合成的牛蛙抗菌肽Palustrin-Ca和Temporin-La对小鼠金葡萄感染创面有明显的抗感染作用并能促进创面的愈合。本研究为牛蛙皮肤抗菌活性肽的多样性提供了依据,同时也为研究多样性的两栖动物皮肤活性肽,发现新型抗菌、抗癌药物前体物质,设计新型活性肽建立了研究方法和参考。
A variety of bio-active substances from amphibian skin that play an important role in defense against invading factors have attracted significant attention of the researchers all over the world. So far, many kinds of proteins and polypeptides have been isolated and characterized from amphibian. Rana catesbeiana originally lived in Rocky Mountain north America, which were introduced into China in 1959, and have raised at a large amount in middle and south of China now. The antibacterial peptides from the skin of Rana catesbeiana play an essential role in amphibian innate immune system and reportedly have stronger killing effect than any other common drugs on, pseudomonas aeruginosa that could cause blind on man, but without problems of drug resistance.
To analyze the diversity and structures of the skin antibacterial peptides of Rana catesbeiana, Rana catesbeiana skin cDNA library was constructed with about 1×106 independent clones. According to GenBank, degenerate primers were designed for antibacterial peptides sequence of Rana catesbeiana based on the conservative region of signal peptide of Rana antibacterial peptides. Genes of antibacterial peptide were amplified from the library by PCR methods. One hundred of cDNA clones were sequenced, and 48 full-length cDNA sequences encoding 24 kinds of antimicrobial peptides precursor were obtained, among which 18 antimicrobial peptides precursor were subordinated by nine known antimicrobial families previously identified in the skins of Ranid frog s:Ranatuerin-1(6),Ranatuerin-2(3), temporin(3),Palustrin(1),Ranatuerin-5 (3),Ranatuerin-7(1),Ranatuerin-8(1)and other six anti-microbial peptides precursor had been newly found antimicrobial peptides which had much lower homology than others known,and were named and divided into three families: Catesbeianin-1(Catesbeianin-1), Ranacyclin(Ranacyclin-Ca,Cb and Cc),temporin (Temporin-La and Lb).The signal and propiece domain(SPD)of the peptides were extremely conserve while mature peptide domain had much more variation, that they were perhaps originated from a common ancestor. It was also supposed that formation of diversity of antimicrobial peptides have relationship with construction of microorganism in bullfrog’s living environment. Transition and transversion between every another nucleotide sequence of nine antimicrobial peptide families were conducted, the results suggested that there were difference substitution patterns in the two domains, perhaps because they suffered from different selective pressure in the evolution.
Seven newly found polypeptides named Temporin-La,Temporin-Lb,Catesbeian in-1,Ranacyclin-Ca, Ranacyclin-Cb,Palustrin-Ca,Temporin-1Ca were synthesized by chemosynthesis methods, Biological activities including antibacterial, hemolytic, hemagglutination, trypsin activity/trypsin-inhabiting,antioxidation, NO release, antitumor, antivirus were assayed and the results were as follows:
(1) For studying the antibacterial activities, Minimum Inhibitory Concentration (MIC) methods were determined, and the results showed that antimicrobial peptides of Temporin family had stronger inhibition activities on staphylococcus aureus and streptococcus suis. The MIC of staphylococcus aureus by Temporin-La was 2.5μg /mL and streptococcus suis by Temporin-Lb was 7.8μg/mL, Palustrin-Ca was 7.8μg/mL.
(2) Detection of hemolytic indicated that hemolytic rates of Catesbeianin-1, Temporin-1Ca, Palustrin-Ca for rabbit red blood cells were 0.8%, 0.30%, 0.25% respectively. Temporin-Lb have strong hemolytic activity with a hemolytic rate amounted to 31.56%, while that of Temporin-La was 1.75%. The C-end of the two peptides were both animated, variation of only two amino acids, Leu at 2, 9 site in Temporin-Lb was substituted with Phe at 2, 9 site. Although both of them were hydrophobic amino acids, their vicariousness may cause different hemolytic activity between the two peptides. Perhaps the differences of amino acids lead to differences of hemolytic activity, which would provide reference for molecular designing of antibacterial peptide.
(3)Detection of activities of trypsin and trypsin inhabitation showed that both Ranacyclin-Ca and Ranacyclin-Cb polypeptides had activities of s trypsin inhabitation. Compared to known trypsin inhibitor, there were two amino acids difference in the motifs of active region. Both of the Ranacyclin-Ca and Ranacyclin-Cb were thermal stability and effective irreversible trypsin inhabitation activity with a Ki value of 7.02×10~(-7)M and 5.6×10~(-7)M respectively.
(4) Erythrocyte agglutination activity detection showed that a lectin of Rana catesbeiana (Temporin-1Ca) with the molecular weight of 1471.8Da, which is smaller than any other known lectin from amphibian, has very low homology with the other lectin. Temporin-1Ca had very high rabbit erythrocyte agglutination activity with the minimum concentration 12.5μg/mL. It was also indicated that Temporin-1Ca had significant bacterial agglutination activity by laser scanning confocal microscope for Staphyloccocus aureus, streptococcus suis, Salmonella, Actinobacillus pleuropneu moniae.
(5) Temporin-Lb and Catesbeianin-1 had stronger elimination ability of DPPH with the rate of 76.50% and 69.47% respectively. Moreover, Palustrin-Ca was able to promote NO releasing. With a concentration of 10μg/mL, Palustrin-Ca can promote 57.45% of the NO release.
(6) Temporin-Lb and Palustrin-Ca had stronger anticancer activities, the IC_(50) of Temporin-Lb to gastric tumor cell SMMC7721 strain was 1.384μg/ mL, as well as the IC_(50) of Palustrin-Ca to the liver tumor cell SGC 7901 strain was 0.95μg/ mL.
(7)Antiviral activity detection showed that At its safe concentration,The inhibition rates of Palustrin-Ca, Catesbeianin-1 and Temporin-La to the SIV were 88.79%, 84.30% and 80.76%,respectively.
By random screening of cDNA library of bullfrog skin, a cDNA sequence of a kind of bradykinin-precurosor of Kininogen-1Ca from Rana catesbeiana were obtained, which had 77% identity with kininogen-1 precursor from Rana pipiens. The bradykinin which encoding RL16 ( RPPGCNPFRIAPASYL ) and RR9(RPPGCNPFR)was detected using biologic function radiomete on the influence of contraction ability of rat ileum muscle, bradykinin could contraction rat ileum muscle significantly. This is the first report of bradykinin in Rana catesbeiana and its cDNA sequence.
Summarizing above results, antimicrobial peptides, trypsin inhibitor, lectin and bradykinin were obtained from the skin of bull frog by random screening of cDNA library of bullfrog skin.
The effects of antibacterial peptide Temporin-La, Temporin-Lb, Palustrin-Ca, Catesbeianin-1 were observed that the antibacterial peptides had effect on microstructure of Staphylococcus aureus and streptococcus suis serotype 2 using transmission electron microscope.Temporin-La and Palustrin-Ca could caused the plasmolysis of Staphylococcus aureus ,while Temporin-Lb and Catesbeianin-1 could cause the bacteria formed a blurring fibrosis of bacterial cell wall between cell wall and cytomembrane. All the four peptides could cause cell wall clearage of streptococcus suis. There were various kinds of different mechanism of antibacterial peptide should be researched.
Temporin-La and Palustrin-Ca with high bacteriostasis activity were studied in treating infected wound in mice skin with Staphylococcus aureus, the number of bacteria in wound, the number of leucocytes, tissue slice, as well as the expression level of vascular endothelial growth factor (VEGF) were determined. Consequently, the results showed that the synthetical antibacterial peptide had significant effects of anti-inflammatory and the healing effect of Palustrin-Ca was better than that of Temporin-La as well as antibiotics treatment group.
The screened peptides studied in this study might become precursors for new drugs developing and provide potential new templates to further explore. In addition, this study provided a reference for the studying of the diversity of bioactive peptides of amphibians, new antibiotic or anticancer drugs developing and new type bioactive peptide designing.
引文
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