人源抗菌肽LL-37在毕赤酵母中的高效表达及其活性检测

根据GenBank CAA86115中的LL-37氨基酸序列, 选择毕赤酵母偏好密码子, 采用SOE方法合成了人源抗菌肽LL-37基因。所合成的LL-37基因全长为141 bp, 并在其N端引入kex2裂解位点, 以保证表达抗菌肽具有天然N端。基因克隆入pPICZa-A质粒, 构建分泌型重组酵母表达载体pPICZa-A-LL-37。pPICZa-A-LL-37经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X-33。PCR鉴定为阳性的酵母转化子经甲醇诱导分泌LL-37于发酵上清液, 其表达量为206 mg/L。表达产物LL-37耐热性强, 在100℃条件下40 min内抗菌活性不变, 煮沸3 h以上仍具有活性。琼脂糖孔穴扩散法检测显示LL-37对多种革兰氏阴性菌和阳性菌均具有很好的抑制活性, 其对金黄色葡萄球菌 CowanⅠ(Staphylococcus aureus)、致病性大肠杆菌K99(Enteropathogenic E.coli)和鸡白痢沙门氏菌(Salmonella pullorum)的最小抑菌浓度(Minimal Inhibitory Concentration, MIC)分别为1.56 mg/mL、3.12 mg/mL和1.56 mg/mL。     

人源抗菌肽LL-37的原核表达及活性鉴定

目的:实现大肠杆菌高效可溶表达人源抗菌肽LL-37。方法:LL-37基因克隆至原核载体pET32a,于大肠杆菌BL21(DE3)中诱导表达。运用相关生物信息学软件分析重组蛋白Trx-LL-37的理化性质、亲/疏水性、蛋白质二级结构及其可溶表达概率。实验还考察了不同诱导温度对重组蛋白可溶表达比例的影响。结果:生物信息学分析显示,Trx-LL-37分子量21.5kD,理论等电点6.3,物理性质稳定,二级结构简单,具有可溶表达倾向。重组蛋白最佳诱导温度为17℃,与37℃相比,可溶表达比例由37.2%提高至50.2%,并且总表达量也提高了5%左右。抑菌结果显示纯化产物对多种常见细菌的生长具有抑制作用。结论:可采用融合方式通过原核系统高效可溶表达LL-37,为LL-37的功能研究打下基础。     

内含肽介导PHB纯化人源抗菌肽LL-37体系的构建

新颖的内含肽介导PHB纯化蛋白体系,是一种高效表达、自动切割、纯化方便、费用低廉的蛋白表达纯化体系,有利于蛋白规模化纯化。本研究选用对原核细胞具有毒害作用的小肽--人源抗菌肽LL-37作为纯化对象,通过基因工程技术,构建内含肽介导PHB纯化人源抗菌肽LL-37体系,并利用该体系纯化LL-37。研究结果表明,本研究构建的内含肽介导PHB纯化人源抗菌肽LL-37体系可高效表达LL-37融合蛋白,利用构建的纯化体系能对目的蛋白进行纯化。     

人源抗菌肽LL-37在毕赤酵母中的高效表达及其活性检测

根据GenBank CAA86115中的LL-37氨基酸序列,选择毕赤酵母偏好密码子,采用SOE 方法合成了人源抗菌肽LL-37基因.所合成的LL-37基因全长为141 bp,并在其N端引入kex2裂解位点,以保证表达抗茵肽具有天然N端.基因克隆入pPICZα-A质粒,构建分泌型重组酵母表达载体pPICZα-A-LL-37.pPICZα-A-LL-37经Sac Ⅰ酶切线性化后电转化导入毕赤酵母菌株X-33.PCR鉴定为阳性的酵母转化子经甲醇诱导分泌LL-37于发酵上清液,其表达量为206mg/L.表达产物LL-37耐热性强,在100℃条件下40 min内抗茵活性不变,煮沸3 h以上仍具有活性.琼脂糖孔穴扩散法检测显示LL-37对多种革兰氏阴性茵和阳性菌均具有很好的抑制活性,其对金黄色葡萄球菌Cowan I (Staphylococcus aureus)、致病性大肠杆菌K99(Enteropathogenic E.coli)和鸡白痢沙门氏菌(Salmonella pullorum)的最小抑菌浓度(Minimal Inhibitory Concentration,MIC)分别为1.56 μg/mL、3.12 μg/mL和1.56 μg/mL.     

微生物源抗菌肽研究概况

抗菌肽是广泛存在于生物体内的一种小分子多肽,具有分子量小、高效、稳定、作用机制独特和不易产生耐药性等特点,对细菌、真菌、寄生虫、病毒以及肿瘤细胞均有抑制作用。介绍了微生物来源,尤其是细菌源抗菌肽的结构特点、生物活性、作用机制及其在感染性疾病中的应用情况。     

重组酵母产人IFN-α2a与LL-37融合蛋白的工艺优化

通过单因素试验探索重组酵母P.pastoris GS115LI产人LL-37基因与IFN-α2a融合蛋白的最佳条件。结果显示,重组酵母菌产蛋白酶的最佳表达条件是利用BMMY为诱导培养基,以接种量OD600=5.5、温度26℃、p H6.0、诱导剂(甲醇)为每隔24 h添加1.0%、振荡速度为200 r/min条件下连续诱导144 h。在最佳培养条件下,发酵液中的LL-37最高抗菌活性达27.9 mm。与初始的发酵条件相比,人LL-37基因与IFN-α2a融合蛋白的产量提高了32.29%。     

抗菌肽LL-37抑制肝癌细胞恶性增殖的转录组分析

抗菌肽LL-37与肿瘤的发生发展密切相关,课题组前期研究发现LL-37能够抑制肝癌细胞恶性增殖。为了给其抑制肝癌发生发展的分子机制研究提供更多的生物学依据,本研究通过高通量RNA测序技术以及生物信息学方法对LL-37作用前后肝癌细胞中的差异表达基因(differentially expressed gene, DEG)进行了分析,共筛选出753个DEG,其中上调的DEG 374个,下调的DEG 379个;进一步对筛选出的DEG进行基因本体论(Gene Ontology, GO)及京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes, KEGG)通路富集分析,并构建DEG编码蛋白互作网络,筛选出10个可能参与LL-37抑制肝癌细胞恶性增殖的潜在关键基因。经分析这些基因均表达下调,且对机体炎症的激活、转运以及肿瘤细胞的增殖、迁移至关重要。以上结果为揭示LL-37在肝癌发生发展中的作用及机制提供了数据基础,为探索肝癌诊断和治疗手段提供了新的思路。     

植物源抗菌肽的研究进展

植物抗菌肽是植物自身合成的能够防御环境中微生物侵害的一类小分子多肽.它们大都是阳离子多肽,有较好的热稳定性.根据作用位点和抗菌机理的不同,植物抗菌肽可分为三大类:第一类通过干扰微生物细胞壁的合成来抑制微生物生长;第二类作用于质膜使其产生穿膜孔洞,从而导致微生物因细胞物质外泄受损;第三类则通过抑制某些细胞器的作用而起到抑菌的效果.本文从抗菌肽的作用机理及其分类等方面对植物源抗菌肽的研究进展进行了综述.     

LL-37-haFGF融合蛋白的结构预测与生物信息学分析

目的:为开发一种多功能蛋白分子,该文采用生物信息学方法对LL-37-haFGF融合蛋白进行结构预测与分析.方法:重叠PCR技术克隆LL-37-haFGF基因,运用生物信息学软件分析LL-37-haFGF编码蛋白序列和结构.结果:LL-37-haFGF融合基因编码215个氨基酸,分子量为23.8 kDa,理论等电点为8.86,利于基因工程表达,结构分析表明其结构有利于多功能活性的保持.结论:融合基因的生物信息学分析与结构预测为研究其活性和作用机制奠定了基础.     

人LL-37与干扰素α2a密码子的优化及其在毕赤酵母中的融合表达

通过密码子优化,在毕赤酵母中高效表达人LL-37与IFN-α2a融合蛋白。先按P.pastoris密码子偏好性对LL-37与IFN-α2a的原始密码子进行了改造,并在二者之间加上Gly Gly Gly Gly Ser的柔性连接接头,人工合成设计的新序列,最后通过p PIC9K载体将其整合入P.pastoris GS115的基因组,构建出重组菌株GS115LI。利用遗传霉素浓度梯度筛选出两株高拷贝的GS115LI1和GS115LI2菌株。对这两株菌经发酵诱导后的发酵液进行SDS-PAGE检测、抗病毒活性检测和抗菌活性检测,证明重组株既能够成功表达出LL-37与IFN-α2a的融合蛋白,而且该融合蛋白成功保留了抗菌肽与干扰素的功能活性。诱导发酵后,融合蛋白的产量可达到819.1 mg/L,经盐析、疏水层析和离子交换层析分离,可得到纯度达97%的融合蛋白产物,回收率可达到46.2%,纯化后产品的效价可达2.6×108 IU/mg。经过密码子优化后,成功实现在毕赤酵母中高效表达出既有LL-37抗菌活性又具有干扰素α2a活性的融合蛋白。     

Prokaryotic selectivity and LPS-neutralizing activity of short antimicrobial peptides designed from the human antimicrobial peptide LL-37

To develop novel antimicrobial peptides (AMPs) with shorter lengths, improved prokaryotic selectivity and retained lipolysaccharide (LPS)-neutralizing activity compared to human cathelicidin AMP, LL-37, a series of amino acid-substituted analogs based on IG-19 (residues 13-31 of LL-37) were synthesized. Among the IG-19 analogs, the analog a4 showed the highest prokaryotic selectivity, but much lower LPS-neutralizing activity compared to parental LL-37. The analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed LPS-neutralizing activity comparable to that of LL-37, but much lesser prokaryotic selectivity. These results indicate that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of LPS-neutralizing activity and prokaryotic selectivity. Furthermore, to increase LPS-neutralizing activity of the analog a4 without a remarkable decrease in prokaryotic selectivity, we synthesized Trp-substituted analogs (a4-W1 and a4-W2), in which Phe(5) or Phe(15) of a4 is replaced by Trp. Despite their same prokaryotic selectivity, a4-W2 displayed much higher LPS-neutralizing activity compared to a4-W1. When compared with parental LL-37, a4-W2 showed retained LPS-neutralizing activity and 2.8-fold enhanced prokaryotic selectivity. These results suggest that the effective site for Trp-substitution when designing novel AMPs with higher LPS-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. Taken together, the analog a4-W2 can serve as a promising template for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection.     

Assessment of antimicrobial peptide LL-37 as a post-exposure therapy to protect against respiratory tularemia in mice

Early activation of the innate immune response is important for protection against infection with Francisella tularensis live vaccine strain (LVS) in mice. The human cathelicidin antimicrobial peptide LL-37 is known to have immunomodulatory properties, and therefore exogenously administered LL-37 may be suitable as an early post-exposure therapy to protect against LVS infection. LL-37 has been evaluated for immunostimulatory activity in uninfected mice and for activity against LVS in macrophage assays and protective efficacy when administered post-challenge in a mouse model of respiratory tularemia. Increased levels of pro-inflammatory cytokine IL-6, chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL1 with increased neutrophil influx into the lungs were observed in uninfected mice after intranasal administration of LL-37. Following LVS challenge, LL-37 administration resulted in increased IL-6, IL-12 p70, IFNγ and MCP-1 production, a slowing of LVS growth in the lung, and a significant extension of mean time to death compared to control mice. However, protection was transient, with the LL-37 treated mice eventually succumbing to infection. As this short course of nasally delivered LL-37 was moderately effective at overcoming the immunosuppressive effects of LVS infection this suggests that a more sustained treatment regimen may be an effective therapy against this pathogen.     

LL-37, the only human member of the cathelicidin family of antimicrobial peptides

Antimicrobial peptides and their precursor molecules form a central part of human and mammalian innate immunity. The underlying genes have been thoroughly investigated and compared for a considerable number of species, allowing for phylogenetic characterization. On the phenotypical side, an ever-increasing number of very varied and distinctive influences of antimicrobial peptides on the innate immune system are reported. The basic biophysical understanding of mammalian antimicrobial peptides, however, is still very limited. This is especially unsatisfactory since knowledge of structural properties will greatly help in the understanding of their immunomodulatory functions. The focus of this review article will be on LL-37, the only cathelicidin-derived antimicrobial peptide found in humans. LL-37 is a 37-residue, amphipathic, helical peptide found throughout the body and has been shown to exhibit a broad spectrum of antimicrobial activity. It is expressed in epithelial cells of the testis, skin, the gastrointestinal tract, and the respiratory tract, and in leukocytes such as monocytes, neutrophils, T cells, NK cells, and B cells. It has been found to have additional defensive roles such as regulating the inflammatory response and chemo-attracting cells of the adaptive immune system to wound or infection sites, binding and neutralizing LPS, and promoting re-epthelialization and wound closure. The article aims to report the known biophysical facts, with an emphasis on structural evidence, and to set them into relation with insights gained on phylogenetically related antimicrobial peptides in other species. The multitude of immuno-functional roles is only outlined. We believe that this review will aid the future work on the biophysical, biochemical and immunological investigations of this highly intriguing molecule.     

Lipopolysaccharide Phosphorylation by the WaaY Kinase Affects the Susceptibility of Escherichia coli to the Human Antimicrobial Peptide LL-37

The human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction.     

LL-37 attenuates inflammatory impairment via mTOR signaling-dependent mitochondrial protection

The human cationic antimicrobial protein LL-37 is a multifunctional host defense peptide with a wide range of immunomodulatory activities. Previous work has shown that LL-37 exerts both pro- and anti-inflammatory effects. The role of mitochondria in the skin inflammatory effects of LL-37 has not been well studied. Therefore, our aim was to investigate the immunomodulatory effect of LL-37 in HaCaT cells and to delineate the underlying mechanisms related to mitochondrial function. Immunohistochemistry results from tissue microarrays showed strong cytoplasmic LL-37 staining in inflammatory cells in chronic dermatic inflammation. Using exogenous LL-37 stimulation and LL-37 knockdown and overexpression, LL-37 was demonstrated to dramatically reduce the mRNA levels and protein secretion of inflammatory cytokines including IL-6, IL-8, IL-1α and tumor necrosis factor-α (TNF-α), which are induced by lipopolysaccharides (LPS). The anti-inflammatory effects of LL-37 are dependent upon its ability to increase mitochondrial biogenesis and to maintain mitochondrial homeostasis. Furthermore, we observed that LL-37 enhances the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2) and mammalian target of rapamycin (mTOR). The mTOR inhibitor rapamycin can neutralize the protective effects of LL-37 on mitochondria. In conclusion, these results suggest that high LL-37 expression levels correlate with chronic skin inflammation; mitochondrial dysfunction occurs in HaCaT cells during inflammation; and LL-37 attenuates inflammatory impairment by stimulating mitochondrial biogenesis and protecting mitochondrial function, which are dependent upon mTOR signaling. These findings provide new insights into targeting mitochondria with LL-37 to prevent skin inflammatory reactions.     

Differential regulation of human cathelicidin LL-37 by free fatty acids and their analogs

LL-37 is the single cathelicidin host defense peptide in humans with direct antimicrobial and immunomodulatory activities. Specific regulation of LL-37 synthesis has emerged as a novel non-antibiotic approach to disease control and prevention. Short-chain fatty acids, and butyrate in particular, were found recently to be strong inducers of LL-37 gene expression without causing inflammation. Here, we further evaluated the LL-37-inducing efficiency of a broad range of saturated free fatty acids and their derivatives in human HT-29 colonic epithelial cells and U-937 monocytic cells by real-time RT-PCR. Surprisingly, we revealed that valerate, hexanoate, and heptanoate with 5–7 carbons are more potent than 4-carbon butyrate in promoting LL-37 gene expression in both cell types. Free fatty acids with longer than 7 or shorter than 4 carbons showed only a marginal effect on LL-37 expression. Studies with a series of fatty acid derivatives with modifications in the aliphatic chain or carboxylic acid group yielded several analogs such as benzyl butyrate, trans-cinnamyl butyrate, glyceryl tributyrate, and phenethyl butyrate with a comparable LL-37-inducing activity to sodium butyrate. On the other hand, although reactive, the anhydride derivatives of short- and medium-chain fatty acids are as potent as their corresponding free acid forms in LL-37 induction. Thus, these newly identified free fatty acids and their analogs with a strong capacity to augment LL-37 synthesis may hold promise as immune boosting dietary supplements for antimicrobial therapy.