Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.     

羊支原体性肺炎两标准株对抗菌药物敏感性的研究

本实验测定了环丙沙星、氧氟沙星、单诺沙星、红霉素、罗红霉素、泰勒菌素、泰妙菌素、四环素等8种药物对羊肺炎支原体两个标准株Y98和Y-goat的体外抑菌浓度以及红霉素与氧氟沙星、泰勒菌素对Y-goat和四环素与氧氟沙星、泰勒菌素对Y98的联合药敏作用。对羊肺炎支原体两个标准株对抗菌药物的敏感性进行了分析。     

Expression of a recombinant crustacean hyperglycemic hormone of the kuruma prawn Penaeus japonicus in methylotrophic yeast Pichia pastoris

ABSTRACT:   Until now, six crustacean hyperglycemic hormones (CHH) designated Pej-SGP-I, -II, -III, -V, -VI and -VII have been characterized in the kuruma prawn Penaeus japonicus . All CHH consist of 72 amino acid residues and have an amidated carboxyl (C)-terminus. In the present study, we expressed Pej-SGP-III in methylotrophic yeast Pichia pastoris in order to obtain a large quantity of recombinant CHH possessing biological activity. A cDNA encoding Pej-SGP-III that had been previously cloned was processed by polymerase chain reaction (PCR) and the resulting product was ligated into an expression vector. Pichia pastoris was transformed with this vector after which a recombinant Pej-SGP-III was expressed having an additional amino acid residue (glycine) at the C-terminus (rPej-SGP-III-Gly), a form considered to be a putative precursor of this hormone. rPej-SGP-III-Gly secreted into the culture medium was purified by reversed-phase HPLC, and amidated using a peptidylglycine alpha-amidating enzyme. The amidated rPej-SGP-III (rPej-SGP-III) showed hyperglycemic activity in in vivo bioassay almost comparable to that of the natural Pej-SGP-III. rPej-SGP-III thus obtained will be a useful tool not only for its physiological study but also for the determination of its 3-D structure.     

A review of antimicrobial peptides: defensins and related cationic peptides

Cationic antimicrobial peptides are present throughout the plant and animal kingdoms and bear striking structural and functional similarities across species lines. They provide primitive, nonspecific means of combating a variety of bacteria, fungi, enveloped viruses, and protozoa. Some are also cytotoxic against host cells, including neoplastic cells. Cationic antimicrobial peptides may play various roles in inflammation and tissue repair. Antimicrobial peptides are found in epithelial tissues regularly exposed to microbial attack as well as in cells whose primary function is defense against potential pathogens. They constitute an important part of the nonoxidative antimicrobial arsenal of leukocytes. They are preformed and/or readily synthesized when the cells are stimulated by exposure to pathogens. They exert their effects directly by inserting into membranes of target cells and forming ion channels which increase membrane permeability; however, antimicrobial peptides can also act as opsonins to facilitate phagocytosis. Resistance to defensins is a virulence factor for organisms such as Salmonella sp. The study of cationic antimicrobial peptides is increasing our understanding of innate immunity, inflammation, and the pathogenesis of genetic diseases such as specific granule disease in humans. Therapeutic applications of antimicrobial peptides are currently under investigation.     

饲粮中添加抗菌肽对肉鸭增重及血清尿素氮、总蛋白水平的影响

240只1日龄肉鸭随机成分4组,每组设3个重复,对照组用基础日粮,试验组在基础日粮上每千克分别添加1、2、3mL抗菌肽制剂。结果显示:1)2、3mL/kg添加量组增重效果显著(P<0.05),3mL/kg添加量组最佳;净肉率升高,达到显著水平(P<0.05),其中主要是胸肌率上升;腹脂率降低但差异不显著(P>0.05);料重比无显著变化(P>0.05)。2)血清总蛋白浓度差异不显著(P>0.05),但尿素氮浓度下降,2、3mL/kg添加量组达显著水平(P<0.05),3mL/kg添加量组最低。     

Biosynthesis of Depsipeptide Mycotoxins in Fusarium

The cyclic hexadepsipeptide enniatin is known as a phytopathogenic compound from Fusaria causing necrosis and wilt. The molecule consists of three alternating residues each of a branched chain amino acid and D-hydroxyisovaleric acid (D-Hiv). Enniatins are synthesized by a 347kDa multienzyme (enniatin synthetase) via a thiol template mechanism. The corresponding gene esyn1 has an open reading frame of 9393 nucleotides and harbours two modules, one responsible for D-hydroxy acid activation and one for L-amino acid activation with an integrated N-methyltransferase domain. Such methyltransferases build an homologous group among N-methyl peptide synthetases. Enniatins are synthesized by step-wise condensation of dipeptidol building blocks in an iterative manner resembling fatty acid synthesis. A key enzyme in enniatin biosynthesis is the NADPH-dependent D-2-hydroxyisovalerate dehydrogenase, that supplies enniatin synthetase with D-Hiv. Enniatins contribute to the wilt toxic character of Fusaria. Virulence was significantly reduced in F. avenaceum after disruption of the esyn1 gene.     

昆虫抗菌肽生物学活性及其应用前景

昆虫抗菌肽是昆虫免疫后存在于血淋巴中的一类活性肽 ,具有广谱的抗菌、抗病毒、抑制肿瘤的生物活性 ,具有很高的应用潜力。本文主要介绍昆虫抗菌肽的类型、生理活性、基因的克隆与表达及在动物科学中的应用前景     

黄芩抗鸡大肠杆菌作用的研究

通过体外和体内两种方法研究黄芩抗鸡大肠杆菌作用.通过体外抑菌作用测定黄芩水煎液对鸡大肠杆菌的最小抑菌浓度（MIC）为2.5g/ml;体内抑菌作用通过对7日龄雏鸡的攻毒与治疗试验来研究,体内治疗药物浓度5g/ml可使90%以上雏鸡获得保护,10g/ml可使100%雏鸡获得保护.以上说明黄芩可以对抗鸡大肠杆菌,可以防治鸡的大肠杆菌病.     

阿莫西林、舒巴坦联用对鸭实验性大肠杆菌病的疗效试验

用试管两倍稀释法测定了阿莫西林、阿莫西林与舒巴坦联合对大肠杆菌的体外抗菌活性,并观察阿莫西林与舒巴坦联合治疗耐阿莫西林大肠杆菌诱发的鸭大肠杆菌病的效果。结果表明:阿莫西林与舒巴坦联合对鸭大肠杆菌产酶菌株体外抗菌活性优于阿莫西林,表现为明显的体外协同作用。阿莫西林与舒巴坦联用治疗耐阿莫西林大肠杆菌诱发的鸭大肠杆菌病,按10mg/kg体重(以阿莫西林量计算)肌肉注射,阿莫西林与舒巴坦联用1∶1和2∶1的有效率、治愈率分别为92.5%、92.5%和85%、80%,均显著高于感染对照组、阿莫西林饮水组及阿莫西林肌肉注射组(P<0.01)。