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.     

Characterization of bovine ileal epithelial cell line for lectin binding,susceptibility to enteric pathogens,and TLR mediated immune responses

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.     

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.     

法氏囊活性肽BP7调节鸡未成熟B细胞的基因表达谱及功能分析

试验旨在探索法氏囊活性肽BP7调节鸡未成熟B细胞的分子基础.利用BP7刺激禽前B淋巴细胞DT40细胞,采用荧光定量PCR(qPCR)检测IgM的mRNA水平,并采用基因芯片分析基因表达谱及其生物学功能.结果 显示,BP7刺激的DT40细胞产生IgM的mRNA水平明显升高.基因芯片分析发现,BP7处理的DT40细胞中共有...     

抗菌肽对抗生素耐药菌株的抑菌活性研究

[目的]研究抗菌肽对抗生素耐药菌株的抑菌活性。[方法]利用抗性平板划线法从腹泻病牛血便中筛选分离出1株耐药菌,通过16S rDNA序列进行鉴定,采用琼脂孔穴扩散法通过梯度盐酸壮观霉素（spectinomycin,Spe⁺）、氨苄青霉素（ampicillin,Amp⁺）、硫酸卡那霉素（kanamycin,Kan⁺）和氯霉素（chloramphenicol,Cm⁺）试验确定该菌药敏特性,并利用1种抗菌肽制剂对该菌株进行药敏试验。[结果]经BLAST比对分析该菌16S rDNA序列,鉴定该耐药菌为科氏葡萄球菌（Staphylococcus cohnii）,此菌对Amp⁺敏感,但对试验中其他抗生素均有耐药性,各梯度抗菌肽对该耐药菌均具有明显的抑菌活性。[结论]抗菌肽能有效抑制耐药科氏葡萄球菌的生长,有望在畜牧生产中代替抗生素使用。     

小肽饲料营养价值及评价方法

小肽一般是指二肽或三肽,小肽饲料就是通过化学或生物方法将本来不适合动物利用的蛋白原料分解,制成含有大量小肽的饲料产品。小肽饲料的营养价值在于小肽的吸收优势和其自身的质量。根据影响小肽营养价值的因素,可以选取小肽的总含量、平均肽链长度、特定小肽的含量、特定氨基酸的含量作为评价小肽饲料营养价值的指标。目前小肽含量的测定方法主要有双缩脲法、福林-酚法、三氯乙酸法、甲醛滴定法、凯氏定氮法、紫外光吸收法、消光系数法、考马斯亮兰法、层析比色法、高效凝胶色谱法、高效液相色谱法和CE-ESI-MS联用法。     

混菌发酵生产富肽蛋白饲料工艺条件的研究

利用枯草芽胞杆菌和产朊假丝酵母菌混合发酵生产富肽蛋白饲料,通过单因素和响应面分析试验对其工艺条件进行了研究,选择接菌比例(枯草芽胞杆菌:产朊假丝酵母)为3:1,发酵36h。最佳发酵条件为:发酵温度33℃,装料量40g,原料初始含水率为62%,拌料水的pH值为自来水的自然pH值。在此条件下测得三氯乙酸可溶性氮(TCA-NSI)含量达到33.61%。