Pathogen_resistant Transgenic Plant of Brassica pekinensis by Transfering Antibacterial Peptide Gene and Its Genetic Stability

The soft rot infected by pathogenic bacterium Erwinia aroideae Holland is one of the three serious diseases of Chinese cabbage ( Brassica pekinensis Rupr.). By constructing vector system of high frequency transformation mediated by Agrobacterium tunefaciens EHA105, anti-bacterial peptide gene with strong bactericidal action to pathogenic bacteria was introduced into Chinese cabbage AB-81 self-bred line and the transgenic plants were obtained. PCR and Southern blotting detection showed that target gene was integrated into plant genome of Chinese cabbage. The tests of bacteriostasis action of the extract from transgenic plants in vitro, and the assay of disease-resistant of transgenic plantlets in the tube and the pot by perfusing inoculation with pathogenic bacteria showed obvious resistance to soft rot. This resistance can be a stable heredity by genetic analysis of generations of transgenic plants self-bred, separation ratio of its R, was 3:1. The resistance to Km and disease of soft rot was still kept in the R-5. These results indicated the possibility of breeding new varieties of anti-soft rot Chinese cabbage by transgenic plants as parents.

Purification and Characterization of an Antibacterial Peptide from Rana Temporaria Chensinensis Skin

The Rana temporaria chensinensis is an amphibian distributing mainly over the north of China. Its skin gland contains a variety of peptides, including antibacterial peptides. One of them, component 1, has been purified from the dried skin of R.temporaria chensinesis using a five step procedure. Purified component 1 has a molecular weight of 5 700 and its amino acid composition shows that it is devoid of disulfide bridges but rich in histine and glycine residues. Moreover, it has been found that it has a broad spectrum of antibacterial activity for Pseuctomonas aeruginosa, Escherichia coli, Bacillus sublilis, S. faccalis and Proteus vulgaris .

The Raman Spectra of the Antibacterial Peptide β-Component of Silkworm · Bombyx Mori · Pupae in Aqueous Solution

he Raman spectra of the antibacterial peptide βcomponent of silkworm· Bombyx Mori· pupae in aqueous solution have been studied. The results show that its mainchain consists of lots of αhelicals and only a few βsheets and βturns. This suggests that the antibacterial peptide βcomponent may be the glycopeptide.

Isolation and Purification of Antibacterial Peptides from Mussel

[ Objective ] This study aimed to extract antibacterial peptides from mussel. [ Method ] Blue mussels were used as raw materials for direct extraction of antibacterial peptides by using O. 5 % acetic acid, and the antibacterial peptides were isolated and purified by Sephacryl S-100 polyacrylamide gel chromatography. The fractions were collected for measurement of antibacterial activity and minimum inhibitory concentration for a variety of bacterial species by filter paper diffusion assay. Molecular weight of the antibacterial peptides was determined by SDS-PAGE. Variation of antibacterial activity of antibacterial peptides was measured at 100 ~C under conditions of different processing time and different pH. [ Result] The O. 5% acetic acid was used for crude extraction of antibacterial peptides as extrac- tion solution and led to relatively high extraction efficiency. By using Sephacryl S-100, the antibacterial peptides could be purified as a single substance. The isola- ted and purified antibacterial peptides of mussel had relatively strong antibacterial properties with molecular weight of 5 908, showing heat-resistance acid-alkaline resistance. [ Conclusion] This study laid the theoretical foundation for large-scale production of antibacterial peotides.

Rational Design of Hybrid Peptides: A Novel Drug Design Approach

Peptides play crucial roles in various physiological and pathological processes. Consequently, the investigation of peptide-based drugs is a highlight in the research and development of new drugs. However, natural peptides are not always ideal choices for clinical application due to their limited number and sometimes cytotoxicity to normal cells. Aiming to gain stronger or specific or novel biological effects and overcome the disadvantages of natural peptides, artificial hybrid peptides have been designed by combining the sequence of two or more different peptides with varied biological functions. Compared to natural peptides, hybrid peptides have shown better therapeutic potentials against bacteria, tumors, and metabolic diseases. In this review, design strategies, structure features and recent development of hybrid peptides are summarized;future directions for the research and development of hybrid peptide drugs are also discussed.

Application of antimicrobial peptides in production life

Antibacterial peptide is a small molecular weight,a substance existing in the biological body,plays a very important role in congenital immune systems.Today,in various antibiotics,the problem of drug resistance has caused people to live an indelible impact.Since the antibacterial peptide has spectral antibacterial properties,it has the characteristics of participating in human natural immunity and no residual,and is now one of the research and development hotspots.Antibacterial peptide is a recognized substance that can replace antibiotics,and people have never stopped,and new types of antimicrobial peptides have also emerged in the Volkswagen Vision.This paper is based on the use of antimicrosis peptides in daily life,briefly describes the effects of antimicrobial peptides in medical,animal husbandry and industrial.

Co-assembled C13-dipeptide hydrogels by Gallic Acid (CA) and epigallocatechin gallate (EGCG) with antibacterial activity

The self-assembled peptide-based hydrogels have been widely utilized in foods.However,the self-assembled hydrogels still have some shortages such as low mechanical properties and hydrogel stabilities.Herein,we designed two co-assembled C_(13)-WS hydrogels conjugated by Gallic Acid(CA)and Epigallocatechin Gallate(EGCG).The introduction of CA and EGCG into C_(13)-WS hydrogels can significantly improve the mechanical properties and hydrogel stabilities.The storage modulus(G’)values of co-assembled C_(13)-WS/CA and C_(13)-WS/EGCG were 13.33 KPa and 16 KPa,respectively.From transmission electron microscope(TEM)results,CA and EGCG contributed to the rearrangement of C_(13)-WS molecules during co-assembly.Moreover,the self-/co-assembled C_(13)-WS hydrogels showed antimicrobial activity against E.coli,P.aeruginosa,and S.epidermidis.The introduction of CA and EGCG significantly enhanced the antibacterial activity of C_(13)-WS hydrogels.Our findings provide a novel strategy for the development of stable supramolecular antibacterial short peptide hydrogels through co-assembly.

Isolation and Preliminary Identification of Antibacterial Active Substances of Antagonistic Actinomycetes against Peach Crown Gall(Agrobacterium tumefaciens)

[ Objective ] The paper was to isolate and preliminarily identify the antibacterial active substances of antagonistic actinomyeete strain G19 obtained from the soil highly affected by peach crown gall （Agrobacterium tumefaciens）. [ Method] The antibacterial substances of antagonistic actinomycete strain G19 were ex- tracted using protein precipitation method, then isolated and purified using high performance liquid chromatography and medium-pressure preparative chromatogra- phy. Its molecular weight was determined by MALDI-TOFMS method, and the related functional groups were verified through chemical color reaction. [ Result] Seven peptide portions were produced from the antibacterial substances of antagonistic actinomycete strain G19 with the molecular weights of 900 - 1 300 Da after isolation and purification. It could be also inferred that it contained Cys, and carried with H2O and Na＋. Color reaction of functional groups verified that the sub- stance was polypeptide containing glycosyl. [ Conclusion] The result provided basis for the final definition of the structure of antibacterial substances in antagonistic actinomycete strain G19.

Crude extract of maggots:Antibacterial effects against Escherichia coli,underlying mechanisms,separation and purification

AIM:To investigate the antibacterial effects of a crude extract of maggots against Escherichia coli(E.coli) and the underlying mechanisms,and to separate and purifythe crude extract of maggots to assess the antibacterial effects of the active ingredients in the crude extract.METHODS:Different concentrations of the crude extract of maggots were incubated with E.coli(O157:H7) and cultured.The optical density(OD) was measured at different time points to plot the OD-T curve.The effects of different concentrations of the crude extract on bacterial membrane permeability were determined by fluorescence probe technique.The effects of different concentrations of the crude extract on plasmid DNA replication were determined by agarose gel electrophoresis.DEAE-Sepharose ion exchange chromatography and Sephacryls-200 HR gel filtration chromatography were used to separate and purify the crude extract of maggots.The molecular weight of proteins in the purified crude extract was determined by SDS-PAGD electrophoresis,and its antibacterial effects were determined by turbidimetric method.RESULTS:The antibacterial effects of the crude extract of maggots at concentrations > 0.5 mg/m L were significant.The antibacterial effects of the crude extract at concentrations of 1.0,1.5 and 2.0 mg/m L did not differ significantly.Fluorescence probe analysis showed that the rate of membrane permeability change was 1223.1% in bacteria incubated with 2 mg/m L of the crude extract,and 1300.0% in those incubated with 80 mg/m L of the crude extract.Plasmid DNA was undetectable in E.coli incubated with 2 and 80 mg/m L of the crude extract.A low molecular weight protein band(about 15 k Da) was detected in the crude extract of maggots and eluent,but not in eluant,from DEAE-Sepharose ion exchange chromatography.The antibacterial effects of the crude extract of maggots and eluent were superior to those of eluant,with the antibacterial effects of eluents being better than those of the crude extract of maggots.Of 24 tubes offiltrates,the antibacterial effects of filtrates in tubes 4,5 and 11 were significantly higher than those of the control.The molecular weight of the protein in filtrates in tubes 4,5 and 11 was about 15 k Da.CONCLUSION:The crude extract of maggots exhibits obvious,dose-dependent antibacterial effects.The crude extract exerts antibacterial effects by changing the bacterial membrane permeability and inhibiting plasmid DNA replication.The prote in that has antibacterial effects in the crude extract of maggots has a molecular weight of about 15 k Da.

Epithelial cell adhesion efficacy of a novel peptide identified by panning on a smooth titanium surface

Epithelial attachment via the basal lamina on the tooth surface provides an important structural defence mechanism against bacterial invasion in combating periodontal disease. However, when considering dental implants, strong epithelial attachment does not exist throughout the titanium-soft tissue interface, making soft tissues more susceptible to peri-implant disease. This study introduced a novel synthetic peptide(A10) to enhance epithelial attachment. A10 was identified from a bacterial peptide display library and synthesized. A10 and protease-activated receptor 4-activating peptide(PAR4-AP, positive control) were immobilized on commercially pure titanium. The peptide-treated titanium showed high epithelial cell migration ability during incubation in platelet-rich plasma. We confirmed the development of dense and expanded BL(stained by Ln5) with pericellular junctions(stained by ZO1) on the peptide-treated titanium surface. In an adhesion assay of epithelial cells on A10-treated titanium, PAR4-AP-treated titanium, bovine root and non-treated titanium, A10-treated titanium and PAR4-AP-treated titanium showed significantly stronger adhesion than non-treated titanium. PAR4-AP-treated titanium showed significantly higher inflammatory cytokine release than non-treated titanium. There was no significant difference in inflammatory cytokine release between A10-treated and non-treated titanium. These results indicated that A10 could induce the adhesion and migration of epithelial cells with low inflammatory cytokine release. This novel peptide has a potentially useful application that could improve clinical outcomes with titanium implants and abutments by reducing or preventing peri-implant disease.

High tolerance to mutations in a Chlamydia trachomatis peptide deformylase loop

AIM:To determine if and how a loop region in the peptide deformylase(PDF)of Chlamydia trachomatis regulates enzyme function. METHODS:Molecular dynamics simulation was used to study a structural model of the chlamydial PDF(cPDF) and predict the temperature factor per residue for the protein backbone atoms.Site-directed mutagenesis was performed to construct cPDF variants.Catalytic properties of the resulting variants were determined by an enzyme assay using formyl-Met-Ala-Ser as a substrate. RESULTS:In silico analysis predicted a significant increase in atomic motion in the DGELV sequence(residues 68-72)of a loop region in a cPDF mutant,which isresistant to PDF inhibitors due to two amino acid substitutions near the active site,as compared to wild-type cPDF.The D68R and D68R/E70R cPDF variants demonstrated significantly increased catalytic efficiency.The E70R mutant showed only slightly decreased efficiency. Although deletion of residues 68-72 resulted in a nearly threefold loss in substrate binding,this deficiency was compensated for by increased catalytic efficiency. CONCLUSION:Movement of the DGELV loop region is involved in a rate-limiting conformational change of the enzyme during catalysis.However,there is no stringent sequence requirement for this region for cPDF enzyme activity.

Identification of a New Peptide Deformylase Gene From Enterococcus faecium and Establishment of a New Screening Model Targeted on PDF for Novel Antibiotics

Objective To identify a new peptide deformylase (PDF) gene (Genebank Accession AY238515) from Enterococcus faecium and to establish a new screening model targeted on PDF. Methods A new PDF gene was identified by BLAST analysis and PCR and was subsequently over-expressed in the prokaryotic expression host E.coli Bl21(DE3). Over-expressed protein was purified for enzymatic assay by metal affinity chromatography and a new screening model was established for novel antibiotics. Result A new PDF gene of Enterococcus faecium was identified successfully. Ten positive samples were picked up from 8000 compound library and the microbial fermentation broth samples. Conclusion A new PDF of gene Enterococcus faecium was first identified and the model had a high efficacy. Positive samples screened may be antibacterial agents of broad spectrum.

Identication of Antimicrobial Peptides Using Chou’s 5 Step Rul

With the advancement in cellular biology,the use of antimicrobial peptides(AMPs)against many drug-resistant pathogens has increased.AMPs have a broad range of activity and can work as antibacterial,antifungal,antiviral,and sometimes even as anticancer peptides.The traditional methods of distinguishing AMPs from non-AMPs are based only on wet-lab experiments.Such experiments are both time-consuming and expensive.With the recent development in bioinformatics more and more researchers are contributing their effort to apply computational models to such problems.This study proposes a prediction algorithm for classifying AMPs and distinguishing between AMPs and non-AMPs.The proposed methodology uses machine learning algorithms to predict such sequences.A dataset was formulated based on 1902 samples of AMPs and 3997 samples of non-AMPs.Machine learning algorithms are trained on a xed number of succinct coefcients retaining sequence and composition information of primary structures.The features are extracted using position relative incidence and statistical moments.System performance is validated via various validation tests including a 10-fold cross-validation approach.An overall accuracy of 95.43%was achieved.A comparison of results with existing methodologies shows that the proposed methodology outperformed existing methodologies in terms of prediction accuracy.

Research progress of plant antimicrobial peptides

Plant antimicrobial peptides are a very large family of antimicrobial peptides,which have strong resistance to various pathogenic microorganisms,especially fungi.With the increasing use of antibiotics,the problems caused by antibiotics,including antibiotic residues and pathogen resistance,are becoming more and more prominent.The research on antimicrobial peptides as new antibiotic substitutes is also a hot spot.This article introduces the action sites and antibacterial mechanisms of several plant antimicrobial peptides,as well as the application of plant antimicrobial peptides in the fields of medicine,agriculture,and food preservation.

Biomimetic AgNPs@antimicrobial peptide/silk fibroin coating for infection-trigger antibacterial capability and enhanced osseointegration

Endowing implant surfaces with combined antibacterial and osteogenic properties by drug-loaded coatings has made great strides,but how to achieve the combined excellence of infection-triggered bactericidal and in vivo-proven osteogenic activities without causing bacterial resistance still remains a formidable challenge.Herein,antimicrobial peptides(AMPs)with osteogenic fragments were designed and complexed on the surface of silver nanoparticle(AgNP)through hydrogen bonding,and the collagen structure-bionic silk fibroin(SF)was applied to carry AgNPs@AMPs to achieve infection-triggered antibacterial and osteointegration.As verified by TEM,AMPs contributed to the dispersion and size-regulation of AgNPs,with a particle size of about 20 nm,and a clear protein corona structure was observed on the particle surface.The release curve of silver ion displayed that the SF-based coating owned sensitive pH-responsive properties.In the antibacterial test against S.aureus for up to 21 days,the antibacterial rate had always remained above 99%.Meanwhile,the underlying mechanism was revealed,originating from the destruction of the bacterial cell membranes and ROS generation.The SF-based coating was conducive to the adhesion,diffusion,and proliferation of bone marrow stem cells(BMSCs)on the surface,and promoted the expression of osteogenic genes and collagen secretion.The in vivo implantation results showed that compared with the untreated Ti implants,SF-based coating enhanced osseointegration at week 4 and 8.Overall,the AgNPs@AMPs-loaded SF-based coating presented the ability to synergistically inhibit bacteria and promote osseointegration,possessing tremendous potential application prospects in bone defects and related-infection treatments.

广西藤茶提取物APS体外抗菌作用研究

目的: 观察从广西藤茶中提取得到的一个单体化合物(简称APS) 的体外抗菌作用。方法: 利用琼脂稀释法对标准金黄色葡萄球菌(S. aureus) 、耐甲氧西林金黄色葡萄球菌( MRSA) 、绿脓杆菌( P.aeruginosa) 、大肠杆菌(E.coli) 、乙型溶血性链球菌( β-hemolytic streptococcus) 、福氏痢疾杆菌( Shigella flexneri) 、白色葡萄球菌(Staphylococcus albus)、奈氏菌(Neisseria)、白色念珠菌(Candida albicans)、枯草杆菌(Bacillus subtilis)进行最低抑菌浓度( MIC) 测定, 并与盐酸黄连素相比较, 利用不同浓度 APS 对标准金黄色葡萄球菌、MRSA、绿脓杆菌进行体外杀菌曲线测定。结果: 对 S. aureus、MRSA、P. aeruginosa、E.coli、β- hemolytic strepto-coccus、Shigella lexneri、Staphylococcus albus、Neisseria、Candida albicans、Bacillus subtilis 的 MIC 值分别为 0.078、0.078、1.25、0.31、2.5、1.25、0.625、2.5、5、1.25mg/mL; 体外杀菌曲线表明, APS 对 S. aureus、MRSA、P. aeruginosa 有直接抑制作用, 且与药物剂量呈正相关。结论: APS 体外具有明显的抗菌作用。

抗菌肽Japonicin II基因真核表达载体的构建

采用SOE法人工设计和合成抗菌肽Japonicin II基因。按正确的阅读框架定向克隆至真核表达载体pPICZαA上,经PCR鉴定及序列分析,所转化的毕赤酵母GS115中含有插入Japonicin II基因的重组质粒pPICZαA-Jap,结果表明成功构建了抗菌肽JaponicinII基因真核表达载体pPICZαA-Jap。

不同浓度的抗菌肽RISE-AP12~对体外培养成骨细胞生物活性的影响

目的通过检测不同浓度的抗菌肽RISE-AP12~对成骨细胞的增殖、迁移、成骨、蛋白合成、碱性磷酸酶活性(alkaline phosphatase,ALP)、细胞传代的影响,探讨抗菌肽局部给药的效应浓度范围。方法将不同浓度的抗菌肽(0、5、10、15、20、25、30 mg/L)加入预培养24 h的MG-63成骨细胞培养液中。孵育1、2、3、4、21 d后,倒置显微镜观察细胞的形态;1、2、3 d后检测其对细胞迁移能力的影响;2 d后检测其对细胞的增殖活性及蛋白合成的影响;4 d后检测其对细胞碱性磷酸酶活性的影响;21 d后检测其对细胞成骨钙结节的影响;细胞连续传代培养3次,检测其对细胞增殖活性的影响,显微镜观察成骨细胞形态。结果 0~20 mg/L抗菌肽对成骨细胞的形态、增殖、迁移、钙结节、蛋白合成、碱性磷酸酶活性及细胞传代均无影响;而25~30mg/L抗菌肽使成骨细胞的形状不规则、死亡增多,使成骨细胞形成钙结节的量减少,抑制细胞的增殖和蛋白合成,促进碱性磷酸酶的活性,细胞传代过程中细胞增殖活性降低、形状改变、死亡增多。结论在抗菌肽抑菌(10 mg/L)、杀菌(20 mg/L)有效浓度内,抗菌肽对细胞的生物活性没有影响,所以10~20 mg/L抗菌肽可用作局部用药。

抗肿瘤肽AP-9的固相合成

建立了抗肿瘤肽AP-9的Fmoc固相全合成方法,产品纯度大于98%。着重研究了Fmoc-Arg(Pbf)-OH与H-Pro-Trt树脂的缩合反应。结果表明,以THF-NMP(1∶1)为溶剂,于30℃微波辐射反应6min,缩合产率达80.2%。

抗菌肽PMAP37基因RT-PCR扩增、序列分析及重组原核表达载体构建

提取猪小肠total RNA,根据已发表PMAP37的基因序列进行RT-PCR扩增,并将扩增产物克隆至pMD19-T simple载体测序;构建pBV220-PMAP37原核表达载体并进行酶切鉴定。结果表明:扩增所得序列516bp,开放阅读框504bp,与目的序列同源性99.6%;pBV220-PMAP37原核表达载体构建成功。