人源杀菌肽LL-37的改良、融合表达及其生物学活性研究
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摘要
杀菌肽(又称抗菌肽、肽抗生素)是近年来发现的生物体基因编码的具有抗微生物活性的小肽,通常是由12-60个氨基酸所组成,分子量＜10KDa,是生物体天然免疫的重要组成部分。目前已证实来自各种哺乳动物的Cathelicidin成员有30多种,而人类唯一的Cathelicidin gene所编码的蛋白质为hCAP-18(human Cationic antimicrobial protein 18),LL-37是hCAP-18 C端的37个氨基酸片段,为分子量4.5kDa的阳离子两性分子α-螺旋抗菌肽,其作用特点在于既不容易导致耐药菌株的产生,又具有广谱抗微生物作用,因而具有极高的应用价值。
研究表明,LL-37在人体内含量较低,而且其杀菌力较猴RL-37等同源杀菌肽的抗菌力低。根据结构与功能(structure-activity relationship,SAR)的研究显示所有α-螺旋杀菌肽的杀菌活性和抗菌谱与肽链中所带正电荷、疏水性氨基酸数目密切相关。通过分子改造增加LL-37肽链的正电荷,来提高其抗菌活性和拮抗内毒素的能力是一种较好的途径。
本研究对LL-37进行分子改造,提高LL-37的正电荷,同时把LL-37基因序列的部分原核细胞稀有密码子替换为原核细胞偏爱密码子,并在其N端加入一段编码负电荷、亲水性强的承载蛋白(Carrier protein molecule,CPM)基因序列,放入载体pET-30a(+),并于工程菌BL21 Star(DE3)中融合表达,使得改建LL-37(reconstructedLL-37,rLL-37)能在原核细胞中高效表达。rLL-37以融合肽形式采用大肠杆菌表达,其引导蛋白中含有FXa酶切位点和6个His的特异亲和纯化标志物,随后从表达的融合蛋白中抽提出包涵体,并经纯化、脱盐、FXa酶切等步骤,当以FXa酶切除N端的引导片段,则获得高纯度的rLL-37,并初步研究其体内外生物学活性。
本研究的主要结果和结论如下:
1、应用蛋白质分析软件(Anthepro 5.0、SWISS-MODEL等),对LL-37多肽进行二维、三维结构及理化特性分析,将LL-37肽链中的部分带负电荷的氨基酸残基替换为中性(带极性基团,易溶)氨基酸残基(Glu_(16)→Gln_(16)、Asp_(26)→Asn_(26)、Glu_(36)→Gln_(36)),构建了改良LL-37多肽(rLL-37),rLL-37的净电荷由LL-37的+5.8提高至+9,并保持了N端亲水极性和C端疏水极性及二维、三维螺旋结构不变。同时将LL-37基因序列的部分原核细胞稀有密码子替换为原核细胞偏爱密码子(GGA替换为GGT、AGA替换为CGT)。
2、设计了一个84bp的DNA序列,该序列编码含28个氨基酸残基的多肽(即承载蛋白质分子,CPM),其pHi 2.7、pH 7.4时净电荷为-6.0。将这一段编码CPM的基因序列放入rLL-37多肽基因序列的N端。
3、采用Touch-Down PCR方法,获得了编码CPM及rLL-37的DNA序列(长约210 bp),将其转入T载体,酶切后置入表达载体pET-30a(+),制备了表达质粒pET-30a(+)-CPM-rLL-37,测序鉴定证实所构建质粒序列正确。
4、质粒pET-30a(+)-CPM-rLL-37于杆菌BL21 star(DE3)中表达,融合蛋白约占全菌蛋白的35%,并被TALON柱芯有效地亲和纯化。融合蛋白以FXa酶切后,rLL-37能被强阳离子交换柱芯Macro-Prep High S有效纯化。通过Folin-酚法测定蛋白质含量共获得rLL-37目的蛋白约3.5mg。
5、用抑菌圈法证实rLL-37杀菌肽对革兰氏阴性菌和革兰氏阳性菌都具有很好的抑菌力。通过微量稀释法测得rLL-37的MIC(Minimal Inhibitive Concentration)、MBC(Minimal Bacteric Concentration),rLL-37对金黄色葡萄球菌(ATCC25923)显出了比氨苄青霉素(AMP)稍好的杀菌活性,对粪肠球菌(ATCC29212)的杀菌活性与AMP相近。
6、通过ELISA实验,证实rLL-37在体外具有与LPS较强的结合力。
7、小鼠体内生物学功能研究,初步证明rLL-37在动物体内无明显的毒副作用,并且在动物体内有减轻炎症的保护作用。
综上所述,我们对LL-37进行改良,其净电荷由+5.8提高至+9,提高了LL-37的正电荷。在制备中过程,将LL-37基因序列中的部分密码子替换为原核细胞偏爱密码子,在rLL-37基因序列的N端加入一段编码带负电荷的CPM基因序列,使得融合蛋白的净电荷降低,利于TALON柱芯亲和纯化,也降低rLL-37多肽对表达宿主菌的损害;由于加入CPM,含rLL-37的融合肽的分子量由8KDa增加到15KDa,从而提高了表达产物的稳定性和表达产量,研究结果证明rLL-37多肽以原核细胞进行融合表达是可行的。通过对rLL-37体内外生物学功能研究,初步证明rLL-37在体外对G~+、G~-菌都具有较好的杀菌力,而且具有与LPS较强的结合力;rLL-37在动物体内无明显的毒副作用,并且对感染小鼠有杀菌和减轻炎症的体内保护效应。从而使得rLL-37以基因工程制备成为可能,为LL-37多肽的进一步深入研究奠定了良好基础。
Bactericidal peptides(anti-bacterial peptide or peptide antibiotics) are small peptides encoded by organism genomic DNA.They have been recognized by their antimicrobial activities in the innate host defense of most living organisms.Most of these peptides consist of 12-60 residues and are less than 10kDa.Antimicrobial peptides play an important role in innate host defense.About 30 cathelicidin members from various mammalian species have been identified.However,only one cathelicidin,hCAP18(human cationic antibacterial protein of 18 kDa) has been found in humans,and its carboxyl-terminal antibacterial peptide,called LL-37,comprises 37 amino acid residues.LL-37 is a a-helical cathelicidin cation peptide owning molecular wieght 4.5kDa.It is showed that LL-37's broad antimicrobial spectrums and not easy to result in drug-resistance.So it is considered to has important applicative value.
According to the research,LL-37 contents is lower in vivo of humanbody,and its bactericidal activity is inferior than monkey homologization bactericidal peptide RL-37. Studies on the basis of structure-activity relationship(SAR) manifest that the bactericidal activity and antibacterial spectrum of all a-helical bactericidal peptides correlate with their own positive charge and the number of hydrophobic amino acid residues.Reconstructing the proteinie sequence to increase positive charge and the number of hydrophobic amino acid residues of LL-37 peptide chain is a better pathway to enhance LL-37 antibacterial activity and antiendotoxic activity.
In this study,we reconstruct the proteinic sequence of human cathelicidin LL-37 in order to increase its positive charge.The rare codons in the LL-37 gene sequence were substituded by the preferred codons of procaryotic cell,and a fragment of carrier protein molecule(CPM) gene order was added to the N termination of it.The reconstructed LL-37(rLL-37) was inserted into vector pET-30a(+),then the rLL-37 was expressed in E.coli.BL21 Star(DE3) by fusion,in order that it was expressed with high efficiency in procaryotic cell.The rLL-37 was expressed in E.coli.by fusion,which was included specital affinity purification labeling sequence of(FXa) and 6 histidine.We obtained purified rLL-37 after the inclusion bodies of expressed rLL-37 were isolated and purified, desalted,Fxa cutting and so on.At last,the biol-activity of rLL-37 in vivo and vitro was studied.The main results are as following:
1.The two-dimensional structure,three-dimensional structure and chemical characters of LL-37 were analyzed by Soft Ware Anthepro 5.0 and SWISS-MODEL.We obtained reconstructed LL-37 after some negative amino acids of human cathelicidin LL-37 were replaced by positive amino acids and the positive charge of LL-37 was increased(Glu_(16), Asp_(26),Glu_(36) of LL-37 were replaced by Gln_(16),Asn_(26),Gin_(36)).The static charge of rLL-37 was increased from +5.8 of LL-37 to 9.0 at pH 7.4.Without changing the N termination hydrophobic characterization,the C termination hydrophibic characterization,the two-dimensional and the three-dimensional of LL-37.At the same time,the rare codons in the LL-37 gene sequence were substituded by the preferred codons of procaryotic cell(GGA was replaced by GGT,AGA was replaced by CGT).
2.To design a fragment of DNA sequence contained 84 bp,a fragment of carrier protein molecule(CPM) gene order was composed,encoded 28 amino-acid residues and its pHi was 2.7 and net change -6.0 at pH 7.4,then adding the CPM gene order own negetive charge to the N termination of the rLL-37.
3.The DNA sequence(210bp) of encoded the CPM and the rLL-37 was obtained successtively by Touch-Down PCR.After the DNA sequence was inserted PMD19-T Simple Vector,and recombined with vector pET-30a(+) again,the expression plasmid pET-30a(+)-CPM-rLL-37 was constructed successtively,which was confirmed by sequence characterization.
4.The expression plasmid pET-30a(+)-CPM-rLL-37 was expressed in E.coli BL21 Star(DE3).The expressed fusion protein accounted for 35%of total bacterio-protein,then the expressed product was purified by using affinity binding chromatography with TALON resins successfully.After being cleaved by thrombin fector Xa,the rLL-37 was purified by using high positive ion exchange column Macro-Prep High S successfully.The objective protein of rLL-37 was obtained totally 3.5 mg by Folin-phenol method determination.
5.The rLL-37 bactericidal peptide was proved byofinhibitory zone to be able to kill both Gram-negative bacteria and Gram-positive bacteria.By the means of MIC and MBC,it was proved that the rLL-37 had the better bactericidal activity than tradition antibiotic AMP killing Staphylococcus aureus and Bacillus coli XL_1-Blue.The rLL-37 had the same bactericidal activity as tradition antibiotic AMP killing Enterococcus faecalis.
6.Through the experimentation of ELISA,the rLL-37 was confirmed that it owned the stronger antiendotoxic activity in vitro.
7.Biological activity of the rLL-37 was confirmed without adverse reaction,and it could protect infected mouse to sterilize and relieve the inflammation in vivo.
In conclusion,human cathelicidin LL-37 was recreconstruct,and the static charge of rLL-37 was increased from +5.8 of LL-37 to 9.0 at pH 7.4,and the rare codons in the LL-37 gone sequence were substituded by the preferred codons of procaryotic cell.To add the CPM gene order own negetive charge to the N termination of the rLL-37,the static charge of fusion protein was decreased,in order that it was purified by using affinity binding chromatography with TALON resins successfully,and the damage of rLL-37 to expressed host bacterium was decreased as well.At the same time,the rLL-37 polypeptide molecular weight was increased from 8 kDa to 15 kDa.Thereby,its expressive stability and expressive production were enhanced.It is feasible to reconstruct human cathelicidin LL-37 and express the protein in bacteria by fusion.Biological activity of the rLL-37 was proved that it is able to kill both Gram-negative bacteria and Gram-positive bacteria,and it owned the stronger antiendotoxic activity in vitro.The rLL-37 was confirmed without adverse reaction, and it could protect infected mouse to sterilize and relieve the inflammation in vivo.It is feasible to produce reconstructed human cathelicidin LL-37 by genetic engineering,which makes it possible to study LL-37 deeply.
研究表明,LL-37在人体内含量较低,而且其杀菌力较猴RL-37等同源杀菌肽的抗菌力低。根据结构与功能(structure-activity relationship,SAR)的研究显示所有α-螺旋杀菌肽的杀菌活性和抗菌谱与肽链中所带正电荷、疏水性氨基酸数目密切相关。通过分子改造增加LL-37肽链的正电荷,来提高其抗菌活性和拮抗内毒素的能力是一种较好的途径。
本研究对LL-37进行分子改造,提高LL-37的正电荷,同时把LL-37基因序列的部分原核细胞稀有密码子替换为原核细胞偏爱密码子,并在其N端加入一段编码负电荷、亲水性强的承载蛋白(Carrier protein molecule,CPM)基因序列,放入载体pET-30a(+),并于工程菌BL21 Star(DE3)中融合表达,使得改建LL-37(reconstructedLL-37,rLL-37)能在原核细胞中高效表达。rLL-37以融合肽形式采用大肠杆菌表达,其引导蛋白中含有FXa酶切位点和6个His的特异亲和纯化标志物,随后从表达的融合蛋白中抽提出包涵体,并经纯化、脱盐、FXa酶切等步骤,当以FXa酶切除N端的引导片段,则获得高纯度的rLL-37,并初步研究其体内外生物学活性。
本研究的主要结果和结论如下:
1、应用蛋白质分析软件(Anthepro 5.0、SWISS-MODEL等),对LL-37多肽进行二维、三维结构及理化特性分析,将LL-37肽链中的部分带负电荷的氨基酸残基替换为中性(带极性基团,易溶)氨基酸残基(Glu_(16)→Gln_(16)、Asp_(26)→Asn_(26)、Glu_(36)→Gln_(36)),构建了改良LL-37多肽(rLL-37),rLL-37的净电荷由LL-37的+5.8提高至+9,并保持了N端亲水极性和C端疏水极性及二维、三维螺旋结构不变。同时将LL-37基因序列的部分原核细胞稀有密码子替换为原核细胞偏爱密码子(GGA替换为GGT、AGA替换为CGT)。
2、设计了一个84bp的DNA序列,该序列编码含28个氨基酸残基的多肽(即承载蛋白质分子,CPM),其pHi 2.7、pH 7.4时净电荷为-6.0。将这一段编码CPM的基因序列放入rLL-37多肽基因序列的N端。
3、采用Touch-Down PCR方法,获得了编码CPM及rLL-37的DNA序列(长约210 bp),将其转入T载体,酶切后置入表达载体pET-30a(+),制备了表达质粒pET-30a(+)-CPM-rLL-37,测序鉴定证实所构建质粒序列正确。
4、质粒pET-30a(+)-CPM-rLL-37于杆菌BL21 star(DE3)中表达,融合蛋白约占全菌蛋白的35%,并被TALON柱芯有效地亲和纯化。融合蛋白以FXa酶切后,rLL-37能被强阳离子交换柱芯Macro-Prep High S有效纯化。通过Folin-酚法测定蛋白质含量共获得rLL-37目的蛋白约3.5mg。
5、用抑菌圈法证实rLL-37杀菌肽对革兰氏阴性菌和革兰氏阳性菌都具有很好的抑菌力。通过微量稀释法测得rLL-37的MIC(Minimal Inhibitive Concentration)、MBC(Minimal Bacteric Concentration),rLL-37对金黄色葡萄球菌(ATCC25923)显出了比氨苄青霉素(AMP)稍好的杀菌活性,对粪肠球菌(ATCC29212)的杀菌活性与AMP相近。
6、通过ELISA实验,证实rLL-37在体外具有与LPS较强的结合力。
7、小鼠体内生物学功能研究,初步证明rLL-37在动物体内无明显的毒副作用,并且在动物体内有减轻炎症的保护作用。
综上所述,我们对LL-37进行改良,其净电荷由+5.8提高至+9,提高了LL-37的正电荷。在制备中过程,将LL-37基因序列中的部分密码子替换为原核细胞偏爱密码子,在rLL-37基因序列的N端加入一段编码带负电荷的CPM基因序列,使得融合蛋白的净电荷降低,利于TALON柱芯亲和纯化,也降低rLL-37多肽对表达宿主菌的损害;由于加入CPM,含rLL-37的融合肽的分子量由8KDa增加到15KDa,从而提高了表达产物的稳定性和表达产量,研究结果证明rLL-37多肽以原核细胞进行融合表达是可行的。通过对rLL-37体内外生物学功能研究,初步证明rLL-37在体外对G~+、G~-菌都具有较好的杀菌力,而且具有与LPS较强的结合力;rLL-37在动物体内无明显的毒副作用,并且对感染小鼠有杀菌和减轻炎症的体内保护效应。从而使得rLL-37以基因工程制备成为可能,为LL-37多肽的进一步深入研究奠定了良好基础。
Bactericidal peptides(anti-bacterial peptide or peptide antibiotics) are small peptides encoded by organism genomic DNA.They have been recognized by their antimicrobial activities in the innate host defense of most living organisms.Most of these peptides consist of 12-60 residues and are less than 10kDa.Antimicrobial peptides play an important role in innate host defense.About 30 cathelicidin members from various mammalian species have been identified.However,only one cathelicidin,hCAP18(human cationic antibacterial protein of 18 kDa) has been found in humans,and its carboxyl-terminal antibacterial peptide,called LL-37,comprises 37 amino acid residues.LL-37 is a a-helical cathelicidin cation peptide owning molecular wieght 4.5kDa.It is showed that LL-37's broad antimicrobial spectrums and not easy to result in drug-resistance.So it is considered to has important applicative value.
According to the research,LL-37 contents is lower in vivo of humanbody,and its bactericidal activity is inferior than monkey homologization bactericidal peptide RL-37. Studies on the basis of structure-activity relationship(SAR) manifest that the bactericidal activity and antibacterial spectrum of all a-helical bactericidal peptides correlate with their own positive charge and the number of hydrophobic amino acid residues.Reconstructing the proteinie sequence to increase positive charge and the number of hydrophobic amino acid residues of LL-37 peptide chain is a better pathway to enhance LL-37 antibacterial activity and antiendotoxic activity.
In this study,we reconstruct the proteinic sequence of human cathelicidin LL-37 in order to increase its positive charge.The rare codons in the LL-37 gene sequence were substituded by the preferred codons of procaryotic cell,and a fragment of carrier protein molecule(CPM) gene order was added to the N termination of it.The reconstructed LL-37(rLL-37) was inserted into vector pET-30a(+),then the rLL-37 was expressed in E.coli.BL21 Star(DE3) by fusion,in order that it was expressed with high efficiency in procaryotic cell.The rLL-37 was expressed in E.coli.by fusion,which was included specital affinity purification labeling sequence of(FXa) and 6 histidine.We obtained purified rLL-37 after the inclusion bodies of expressed rLL-37 were isolated and purified, desalted,Fxa cutting and so on.At last,the biol-activity of rLL-37 in vivo and vitro was studied.The main results are as following:
1.The two-dimensional structure,three-dimensional structure and chemical characters of LL-37 were analyzed by Soft Ware Anthepro 5.0 and SWISS-MODEL.We obtained reconstructed LL-37 after some negative amino acids of human cathelicidin LL-37 were replaced by positive amino acids and the positive charge of LL-37 was increased(Glu_(16), Asp_(26),Glu_(36) of LL-37 were replaced by Gln_(16),Asn_(26),Gin_(36)).The static charge of rLL-37 was increased from +5.8 of LL-37 to 9.0 at pH 7.4.Without changing the N termination hydrophobic characterization,the C termination hydrophibic characterization,the two-dimensional and the three-dimensional of LL-37.At the same time,the rare codons in the LL-37 gene sequence were substituded by the preferred codons of procaryotic cell(GGA was replaced by GGT,AGA was replaced by CGT).
2.To design a fragment of DNA sequence contained 84 bp,a fragment of carrier protein molecule(CPM) gene order was composed,encoded 28 amino-acid residues and its pHi was 2.7 and net change -6.0 at pH 7.4,then adding the CPM gene order own negetive charge to the N termination of the rLL-37.
3.The DNA sequence(210bp) of encoded the CPM and the rLL-37 was obtained successtively by Touch-Down PCR.After the DNA sequence was inserted PMD19-T Simple Vector,and recombined with vector pET-30a(+) again,the expression plasmid pET-30a(+)-CPM-rLL-37 was constructed successtively,which was confirmed by sequence characterization.
4.The expression plasmid pET-30a(+)-CPM-rLL-37 was expressed in E.coli BL21 Star(DE3).The expressed fusion protein accounted for 35%of total bacterio-protein,then the expressed product was purified by using affinity binding chromatography with TALON resins successfully.After being cleaved by thrombin fector Xa,the rLL-37 was purified by using high positive ion exchange column Macro-Prep High S successfully.The objective protein of rLL-37 was obtained totally 3.5 mg by Folin-phenol method determination.
5.The rLL-37 bactericidal peptide was proved byofinhibitory zone to be able to kill both Gram-negative bacteria and Gram-positive bacteria.By the means of MIC and MBC,it was proved that the rLL-37 had the better bactericidal activity than tradition antibiotic AMP killing Staphylococcus aureus and Bacillus coli XL_1-Blue.The rLL-37 had the same bactericidal activity as tradition antibiotic AMP killing Enterococcus faecalis.
6.Through the experimentation of ELISA,the rLL-37 was confirmed that it owned the stronger antiendotoxic activity in vitro.
7.Biological activity of the rLL-37 was confirmed without adverse reaction,and it could protect infected mouse to sterilize and relieve the inflammation in vivo.
In conclusion,human cathelicidin LL-37 was recreconstruct,and the static charge of rLL-37 was increased from +5.8 of LL-37 to 9.0 at pH 7.4,and the rare codons in the LL-37 gone sequence were substituded by the preferred codons of procaryotic cell.To add the CPM gene order own negetive charge to the N termination of the rLL-37,the static charge of fusion protein was decreased,in order that it was purified by using affinity binding chromatography with TALON resins successfully,and the damage of rLL-37 to expressed host bacterium was decreased as well.At the same time,the rLL-37 polypeptide molecular weight was increased from 8 kDa to 15 kDa.Thereby,its expressive stability and expressive production were enhanced.It is feasible to reconstruct human cathelicidin LL-37 and express the protein in bacteria by fusion.Biological activity of the rLL-37 was proved that it is able to kill both Gram-negative bacteria and Gram-positive bacteria,and it owned the stronger antiendotoxic activity in vitro.The rLL-37 was confirmed without adverse reaction, and it could protect infected mouse to sterilize and relieve the inflammation in vivo.It is feasible to produce reconstructed human cathelicidin LL-37 by genetic engineering,which makes it possible to study LL-37 deeply.
引文
[1]Hase K,Murakami M,Iimura M,et al.Expression of LL-37 by human gastric epithelial cells as a potential host defense mechanism against Helicobacter pylori[J].Gastroenterology,2003,125(6):1613-1625.
[2]Lehrer RI,Ganz T.Cathelicidins:a family of endogenous antimicrobial peptides[J].Curt Opin Hematol,2002,9(1):18-22.
[3]Hase K,Eckmann L,John D,et al.Cell differentiation is a key determinant of Cathelicidins LL-37/human cationic antimicrobial protein 18 expression by human colon epithelium[J].Infect-Immun,2002,70(2):953-963.
[4]Niyonsaba F,Iwabuchi K,Someya A,et al.A cathelicidins family of human antibacterial peptide LL-37 induces mast cell chemotaxis[J].Immunology,2002,106(1):20-26.
[5]Nagaoka I,Kuwahara AK,Tamura H,et al.Augmentation of the bactericidal activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by amino acid substitutions[J].Inflamm Res,2005,54(2):66-73.
[6]袁榴娣,窦非,梁玉璞,等.含有FXa切割位点的抗菌肽X在大肠杆菌中的融合表达.生物工程学报[J],2000,16(3):411-413.
[7]Nizet V,Ohtake T,Lauth X,et al.Innate antimicrobial peptide protects the skin from invasive bacterial infection[J].Nature,2001,22;414(6862):454-457.
[8]窦非,谢维,董雪呤,等.抗菌肽CMIV末端结构对其活性的影响.中国科学(C辑)[J],2000,30(1):59-64.
[9]Dawn ME,Bowdish DJ,Davidson MG,et al.Immunomodulatory Activities of Small Host Defense Peptides[J].Antimicrob Agents Chemother,2005,49(11):1727-1732.
[10]Hancock REW,Diamond G.The role of cationic antimicrobial peptides in innate host defences[J].Trends Microbiol,2000,8(9):402-410.
[11]Susanne SB,Andreas S,Robert B,et al.Increased levels of antimierobial in tracheal of newborn infants during infection[J].Am J Respir Crit Care Med,2002,165(7):992-995.
[12]Sorensen OE,Follin P,Johnsen AH,et al.Human cathelicidin,hCAP-18,is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3[J]. Blood,2001,97(12):3951-3959.
[13]Henzler KA,Martinez GV,Brown MF,et al.Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37[J].Biochemistry,2004,43(26):8459-69.
[14]Bals R,Lang C,Weiner DJ,et al.Rhesus monkey(Macaca mulatta) mucosal antimicrobial peptides are close homologues of human molecules[J].Clin Diagn Lab Immunol,2001,8(2):370-375.
[15]Zhao C,Nguyen T,Boo LM,et al.RL-37,an alpha-helical antimicrobial peptide of the rhesus monkey[J].Antimicrob Agents Chernother,2001,45(10):2695-2702.
[16]Nagaoka I,Hirota S,Niyonsaba F,et al.Augmentation of the lipopolysaccharide -neutralizing activities of human cathelicidin CAP18/LL-37-derived antimi-crobial peptides by replacement with hydrophobic and cationic amino acid residues[J].Clin Diagn Lab Immunol,2002,9(5):972-982.
[17]饶贤才,金晓琳,胡晓梅等,一个承载分子的设计与肽抗生素hPAB-β的高效表达[J].第三军医大学学报,2002,24(4):389-392.
[18]Nell M,Tjabringa G,Wafelman A,et al.Development of novel LL-37 derived antimicrobial peptides with LPS and LTA neutralizing and antimicrobial activities for therapeutic application[J].Peptides,2006,27(4):649-660.
[19]胡福泉,肽抗生素研究进展及其应用前景[J]。微生物学杂志,2003,23(2):59。
[20]Nagaoka I,Hirota S,Niyonsaba F,et al.Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14(+) cells[J].Immunol,2001,167(6):3329.
[21]Fukumoto K,Nagaoka I,Yamataka A,et al.Effect of antibacterial cathelicidin peptide CAP 18/LL-37 on sepsis in neonatal rats[J].Pediatr Surg Int,2005,21(1):20.
[22]Scott MG,Davidson DJ,Gold MR,et al.The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses[J].Immunol,2002,169(7):3883.
[23]Gennaro R,Zanetti M.Structural features and biological activities of the cathelicidinderived antimicrobial peptides[J].Biopolymers,2000,55(1):31.
[24]Johansson J,Gudmundsson GH,Rottenberg ME,et al.Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37[J].Biol Chem, 1998,273(6):3718.
[25]Oren Z,Lerman JC,Gudmundsson GH,et al.Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes:relevance to the molecular basis for its non-cell-selective activity[J].Bioehem,1999,341(3):501.
[26]Zanetti M,Gennaro R,Romeo D,et al.Cathelicidins:a novel protein family with a common proregion and a variable C-terminal antimicrobial domain[J].FEBS Lett,1995,374:1.
[27]Tossi A,Sandri L,Giangaspero A,et al.Amphipathic,alpha-helical antimicrobial peptides[J].Biopolymers,2000,55(1):4.
[28]Li X,Li Y,Han H,et al.Solution Structures of Human LL-37 Fragrnentsand NMR-Based Identification of a Minimal Membrane-Targeting Antimicrobial and Anticancer Region.[J].Am Chem Soc 2006,128(17):5776-85.
[29]Koczulla R,von Degenfeld G,Kupatt C,et al.An angiogenic role for the human peptide antibiotic LL-37/hCAP-18[J].J Clin Invest,2003,111(11):1665.
[30]Tjabringa G,Aarbiou J,ninaber D,et al.The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor[J].Immunol.2003 Dec 15;171(12):6690-6696.
[31]Zanetti M.Cathelicidins,multifunctional peptides of the innate immunity[J].Leukoc Biol 2004;75:39-48.
[32]Koczulla R,Von D,Kupatt C,et al.An angiogenic role for the human peptide antibiotic LL-37/hCAP-18[J].Clin Invest.2003 Jun;111(11):1665-1672.
[33]Davidson D,Currie A,Reid G,et al.The cationic antimicrobial peptide LL-37modulates dendritic cell differentiation and dendritic cell-induced T cell polarization[J].Immunol.2004 Jan 15;172(2):1146-1156.
[34]Schaller B,Schulze A,et al.Increased levels of antimicrobial in tracheal of newborn infants during infection[J].Am J Respir Crit Care Med.2002 Apr 1;165(7):992-995.
[35]Howell M,Jones J,Kisich K,et al.Selective killing of vaccinia virus by LL-37:implications for eczema vaccinatum[J].Immunol.2004 Feb 1;172(3):1763-1767.
[36]Yang Y,Zheng G,Li G,et al.Expression of LL-37/hCAP-18 gene in human leukemia cells[J].Leuk Res.2003 Oct;27(10):947-950.
[1]Zanetti M.Cathelicidins,multifunctional peptides of the innate immunity[J].Leukoc Biol 2004;75:39-48.
[2]Dorsehner RA;Pestonjamasp VK;Tamakuwala S et al.Cutaneous injury induces the release of cathelieidin antimicrobial peptides active against group A Streptococcus[J].Invest Dermatol,2001;117(1):91.
[3]杨云霞,王伯瑶等。抗菌肽LL-37作用研究进展[J],四川生理科学杂志,2003,25(2):56-59.
[4]Nagaoka I,Hirota S,Niyonsaba F,et al.Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14(+) cells[J].J Immunol,2001,167(6):3329.
[5]Fukumoto K,Nagaoka I,Yamataka A,et al.Effect of antibacterial cathelicidin peptide CAP18/LL-37 on sepsis in neonatal rats[J].Pediatr Surg Int,2005,21(1):20.
[6]Scott MG,Davidson DJ,Gold MR,et al.The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses[J].Immunol,2002,169(7):3883.
[7]Nagaoka I,Hirota S,Niyonsaba F,et al.Augmentation of the lipopolysaccharideneutralizing activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by replacement with hydrophobic and cationic amino acid residues[J].Clin Diagn Lab Immunol,2002,9(5):972.
[8]De Yang,Chen Q,Schmidt AP,et al.LL-37,the neutrophil granule and epithelial cell-derived cathelicidin,utilizesformyl peptide receptor-like 1(FPRL1) as a receptor tochemoattract human peripheral blood neutrophils,monocytes,and T cells[J].Exp Med,2000,192(7):1069.
[9]Howell M,Jones J,K.isich K,et al.Selective killing of vaccinia virus by LL-37:implications for eczema vaccinatum[J].Immunol.2004 Feb 1;172(3):1763-1767.
[10]Sorensen OE,Follin P,Johnsen AH,et al.Human cathelicidin,hCAP-18,is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3[J].Blood,2001,97(12):3951.
[11]Davidson D,Currie A,Reid G,et al.The cationic antimicrobial peptide LL-37 modulates dendritic cell differentiation and dendritic cell-induced T cell polarization[J].Immunol.2004 Jan 15;172(2):1146-1156.
[12]Henzler-Wildman KA,Martinez GV,et al.Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37[J].Biochemistry,2004,43(26):8459.
[13]Fukumoto K,Nagaoka I,Yamataka A,et al.Effect of antibacterial cathelicidin peptide CAP18/LL-37 on sepsis in neonatal rats[J].,Pediatr Surg Int,2005,21(1):20.
[14]Zhao C,Nguyen T,Boo LM,et al.RL-37,an alpha-helical antimicrobial peptide of the rhesus monkey[J].Antimierob Agents Chemother,2001,45(10):2695.
[15]Dawn ME,Bowdish DJ,Davidson MG,et al.Immunomodulatory Activities of Small Host Defense Peptides[J].Antimicrob Agents Chemother,2005,49(11):1727-1732.
[16]Nell M,Tjabringa G,Wafelman A,et al.Development of novel LL-37 derived antimicrobial peptides with LPS and LTA neutralizing and antimicrobial activities for therapeutic application[J].Peptides,2006,27(4):649-660.
[17]Tossi A,Sandri L,Giangaspero A,et al.Amphipathic,alpha-helical antimicrobial peptides[J].Biopolymers,2000,55(1):4.
[18]Niyonsaba F,Iwabuchi K,Someya A,et al.A cathelicidin family of human antibacterial peptide LL-37 induces mast cell chemotaxis[J].Immunology,2002,106(1):20.
[19]Li X,Li Y,Hart H,et al.Solution Structures of Human LL-37 Fragmentsand NMR-Based Identification of a Minimal Membrane-Targeting Antimicrobial and Anticancer Region.[J].Am Chem Soc 2006,128(17):5776-85.
[20]窦非,谢维,董雪吟等。抗菌肽CMIV末端结构对其活性的影响[J]。中国科学(C 辑),2000,30(1):59。
[21]Nagaoka I,Kuwahara-Arai K,Tamura H,et al.Augmentation of the bactericidal activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by amino acid substitutions[J].Inflamm Res,2005,54(2):66.
[22]Bals R,Lang C,Weiner DJ,et al.Rhesus monkey(Macaca mulatta) mucosal antimicrobial peptides are close homologues of human molecules[J].Clin Diagn Lab Immunol,2001,8(2):370.
[23]Nizet V,Ohtake T,Lauth X,et al.Innate antimicrobial peptide protects the skin from invasive bacterial infection[J].Nature,2001,414(6862):454.
[24]Koczulla R,von Degenfeld G,Kupatt C,et al.An angiogenic role for the human peptide antibiotic LL-37/hCAP-18[J].J Clin Invest,2003,111(11):1665.
[25]胡福泉。肽抗生素研究进展及其应用前景[J]。微生物学杂志,2003,23(2):59.
[2]Lehrer RI,Ganz T.Cathelicidins:a family of endogenous antimicrobial peptides[J].Curt Opin Hematol,2002,9(1):18-22.
[3]Hase K,Eckmann L,John D,et al.Cell differentiation is a key determinant of Cathelicidins LL-37/human cationic antimicrobial protein 18 expression by human colon epithelium[J].Infect-Immun,2002,70(2):953-963.
[4]Niyonsaba F,Iwabuchi K,Someya A,et al.A cathelicidins family of human antibacterial peptide LL-37 induces mast cell chemotaxis[J].Immunology,2002,106(1):20-26.
[5]Nagaoka I,Kuwahara AK,Tamura H,et al.Augmentation of the bactericidal activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by amino acid substitutions[J].Inflamm Res,2005,54(2):66-73.
[6]袁榴娣,窦非,梁玉璞,等.含有FXa切割位点的抗菌肽X在大肠杆菌中的融合表达.生物工程学报[J],2000,16(3):411-413.
[7]Nizet V,Ohtake T,Lauth X,et al.Innate antimicrobial peptide protects the skin from invasive bacterial infection[J].Nature,2001,22;414(6862):454-457.
[8]窦非,谢维,董雪呤,等.抗菌肽CMIV末端结构对其活性的影响.中国科学(C辑)[J],2000,30(1):59-64.
[9]Dawn ME,Bowdish DJ,Davidson MG,et al.Immunomodulatory Activities of Small Host Defense Peptides[J].Antimicrob Agents Chemother,2005,49(11):1727-1732.
[10]Hancock REW,Diamond G.The role of cationic antimicrobial peptides in innate host defences[J].Trends Microbiol,2000,8(9):402-410.
[11]Susanne SB,Andreas S,Robert B,et al.Increased levels of antimierobial in tracheal of newborn infants during infection[J].Am J Respir Crit Care Med,2002,165(7):992-995.
[12]Sorensen OE,Follin P,Johnsen AH,et al.Human cathelicidin,hCAP-18,is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3[J]. Blood,2001,97(12):3951-3959.
[13]Henzler KA,Martinez GV,Brown MF,et al.Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37[J].Biochemistry,2004,43(26):8459-69.
[14]Bals R,Lang C,Weiner DJ,et al.Rhesus monkey(Macaca mulatta) mucosal antimicrobial peptides are close homologues of human molecules[J].Clin Diagn Lab Immunol,2001,8(2):370-375.
[15]Zhao C,Nguyen T,Boo LM,et al.RL-37,an alpha-helical antimicrobial peptide of the rhesus monkey[J].Antimicrob Agents Chernother,2001,45(10):2695-2702.
[16]Nagaoka I,Hirota S,Niyonsaba F,et al.Augmentation of the lipopolysaccharide -neutralizing activities of human cathelicidin CAP18/LL-37-derived antimi-crobial peptides by replacement with hydrophobic and cationic amino acid residues[J].Clin Diagn Lab Immunol,2002,9(5):972-982.
[17]饶贤才,金晓琳,胡晓梅等,一个承载分子的设计与肽抗生素hPAB-β的高效表达[J].第三军医大学学报,2002,24(4):389-392.
[18]Nell M,Tjabringa G,Wafelman A,et al.Development of novel LL-37 derived antimicrobial peptides with LPS and LTA neutralizing and antimicrobial activities for therapeutic application[J].Peptides,2006,27(4):649-660.
[19]胡福泉,肽抗生素研究进展及其应用前景[J]。微生物学杂志,2003,23(2):59。
[20]Nagaoka I,Hirota S,Niyonsaba F,et al.Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14(+) cells[J].Immunol,2001,167(6):3329.
[21]Fukumoto K,Nagaoka I,Yamataka A,et al.Effect of antibacterial cathelicidin peptide CAP 18/LL-37 on sepsis in neonatal rats[J].Pediatr Surg Int,2005,21(1):20.
[22]Scott MG,Davidson DJ,Gold MR,et al.The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses[J].Immunol,2002,169(7):3883.
[23]Gennaro R,Zanetti M.Structural features and biological activities of the cathelicidinderived antimicrobial peptides[J].Biopolymers,2000,55(1):31.
[24]Johansson J,Gudmundsson GH,Rottenberg ME,et al.Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37[J].Biol Chem, 1998,273(6):3718.
[25]Oren Z,Lerman JC,Gudmundsson GH,et al.Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes:relevance to the molecular basis for its non-cell-selective activity[J].Bioehem,1999,341(3):501.
[26]Zanetti M,Gennaro R,Romeo D,et al.Cathelicidins:a novel protein family with a common proregion and a variable C-terminal antimicrobial domain[J].FEBS Lett,1995,374:1.
[27]Tossi A,Sandri L,Giangaspero A,et al.Amphipathic,alpha-helical antimicrobial peptides[J].Biopolymers,2000,55(1):4.
[28]Li X,Li Y,Han H,et al.Solution Structures of Human LL-37 Fragrnentsand NMR-Based Identification of a Minimal Membrane-Targeting Antimicrobial and Anticancer Region.[J].Am Chem Soc 2006,128(17):5776-85.
[29]Koczulla R,von Degenfeld G,Kupatt C,et al.An angiogenic role for the human peptide antibiotic LL-37/hCAP-18[J].J Clin Invest,2003,111(11):1665.
[30]Tjabringa G,Aarbiou J,ninaber D,et al.The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor[J].Immunol.2003 Dec 15;171(12):6690-6696.
[31]Zanetti M.Cathelicidins,multifunctional peptides of the innate immunity[J].Leukoc Biol 2004;75:39-48.
[32]Koczulla R,Von D,Kupatt C,et al.An angiogenic role for the human peptide antibiotic LL-37/hCAP-18[J].Clin Invest.2003 Jun;111(11):1665-1672.
[33]Davidson D,Currie A,Reid G,et al.The cationic antimicrobial peptide LL-37modulates dendritic cell differentiation and dendritic cell-induced T cell polarization[J].Immunol.2004 Jan 15;172(2):1146-1156.
[34]Schaller B,Schulze A,et al.Increased levels of antimicrobial in tracheal of newborn infants during infection[J].Am J Respir Crit Care Med.2002 Apr 1;165(7):992-995.
[35]Howell M,Jones J,Kisich K,et al.Selective killing of vaccinia virus by LL-37:implications for eczema vaccinatum[J].Immunol.2004 Feb 1;172(3):1763-1767.
[36]Yang Y,Zheng G,Li G,et al.Expression of LL-37/hCAP-18 gene in human leukemia cells[J].Leuk Res.2003 Oct;27(10):947-950.
[1]Zanetti M.Cathelicidins,multifunctional peptides of the innate immunity[J].Leukoc Biol 2004;75:39-48.
[2]Dorsehner RA;Pestonjamasp VK;Tamakuwala S et al.Cutaneous injury induces the release of cathelieidin antimicrobial peptides active against group A Streptococcus[J].Invest Dermatol,2001;117(1):91.
[3]杨云霞,王伯瑶等。抗菌肽LL-37作用研究进展[J],四川生理科学杂志,2003,25(2):56-59.
[4]Nagaoka I,Hirota S,Niyonsaba F,et al.Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14(+) cells[J].J Immunol,2001,167(6):3329.
[5]Fukumoto K,Nagaoka I,Yamataka A,et al.Effect of antibacterial cathelicidin peptide CAP18/LL-37 on sepsis in neonatal rats[J].Pediatr Surg Int,2005,21(1):20.
[6]Scott MG,Davidson DJ,Gold MR,et al.The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses[J].Immunol,2002,169(7):3883.
[7]Nagaoka I,Hirota S,Niyonsaba F,et al.Augmentation of the lipopolysaccharideneutralizing activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by replacement with hydrophobic and cationic amino acid residues[J].Clin Diagn Lab Immunol,2002,9(5):972.
[8]De Yang,Chen Q,Schmidt AP,et al.LL-37,the neutrophil granule and epithelial cell-derived cathelicidin,utilizesformyl peptide receptor-like 1(FPRL1) as a receptor tochemoattract human peripheral blood neutrophils,monocytes,and T cells[J].Exp Med,2000,192(7):1069.
[9]Howell M,Jones J,K.isich K,et al.Selective killing of vaccinia virus by LL-37:implications for eczema vaccinatum[J].Immunol.2004 Feb 1;172(3):1763-1767.
[10]Sorensen OE,Follin P,Johnsen AH,et al.Human cathelicidin,hCAP-18,is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3[J].Blood,2001,97(12):3951.
[11]Davidson D,Currie A,Reid G,et al.The cationic antimicrobial peptide LL-37 modulates dendritic cell differentiation and dendritic cell-induced T cell polarization[J].Immunol.2004 Jan 15;172(2):1146-1156.
[12]Henzler-Wildman KA,Martinez GV,et al.Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37[J].Biochemistry,2004,43(26):8459.
[13]Fukumoto K,Nagaoka I,Yamataka A,et al.Effect of antibacterial cathelicidin peptide CAP18/LL-37 on sepsis in neonatal rats[J].,Pediatr Surg Int,2005,21(1):20.
[14]Zhao C,Nguyen T,Boo LM,et al.RL-37,an alpha-helical antimicrobial peptide of the rhesus monkey[J].Antimierob Agents Chemother,2001,45(10):2695.
[15]Dawn ME,Bowdish DJ,Davidson MG,et al.Immunomodulatory Activities of Small Host Defense Peptides[J].Antimicrob Agents Chemother,2005,49(11):1727-1732.
[16]Nell M,Tjabringa G,Wafelman A,et al.Development of novel LL-37 derived antimicrobial peptides with LPS and LTA neutralizing and antimicrobial activities for therapeutic application[J].Peptides,2006,27(4):649-660.
[17]Tossi A,Sandri L,Giangaspero A,et al.Amphipathic,alpha-helical antimicrobial peptides[J].Biopolymers,2000,55(1):4.
[18]Niyonsaba F,Iwabuchi K,Someya A,et al.A cathelicidin family of human antibacterial peptide LL-37 induces mast cell chemotaxis[J].Immunology,2002,106(1):20.
[19]Li X,Li Y,Hart H,et al.Solution Structures of Human LL-37 Fragmentsand NMR-Based Identification of a Minimal Membrane-Targeting Antimicrobial and Anticancer Region.[J].Am Chem Soc 2006,128(17):5776-85.
[20]窦非,谢维,董雪吟等。抗菌肽CMIV末端结构对其活性的影响[J]。中国科学(C 辑),2000,30(1):59。
[21]Nagaoka I,Kuwahara-Arai K,Tamura H,et al.Augmentation of the bactericidal activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by amino acid substitutions[J].Inflamm Res,2005,54(2):66.
[22]Bals R,Lang C,Weiner DJ,et al.Rhesus monkey(Macaca mulatta) mucosal antimicrobial peptides are close homologues of human molecules[J].Clin Diagn Lab Immunol,2001,8(2):370.
[23]Nizet V,Ohtake T,Lauth X,et al.Innate antimicrobial peptide protects the skin from invasive bacterial infection[J].Nature,2001,414(6862):454.
[24]Koczulla R,von Degenfeld G,Kupatt C,et al.An angiogenic role for the human peptide antibiotic LL-37/hCAP-18[J].J Clin Invest,2003,111(11):1665.
[25]胡福泉。肽抗生素研究进展及其应用前景[J]。微生物学杂志,2003,23(2):59.