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.     

The role of ear environment in postharvest susceptibility of maize to toxigenic Aspergillus flavus

A kernel screening assay (KSA) was used to assess the genetic and environmental effects on the vulnerability of maize to aflatoxin accumulation. Kernels of 26 inbred lines that had been grown in seven environments, and 190 lines of the Intermated B73xMo17 (IBM) population grown in one location in the United States, were inoculated with a toxigenic strain of A. flavus and incubated in the dark at 30°C for 6 days. Percent kernel colonization (PKC), sporulation and aflatoxin were influenced by the maize genotypes (G), the location (“ear environment” or E) and the GxE interactions. Overall, low broad‐sense heritabilities were observed for PKC, sporulation and aflatoxin. PKC was significantly correlated with sporulation in all environments. Aflatoxin was positively correlated with colonization for two and with sporulation for all ear environments. Higher grain sulphur or magnesium in IBM was associated with less colonization or aflatoxin. Postharvest susceptibility of maize to aflatoxin is thus influenced by factors that are modulated by the ear environment. In a KSA, sporulation could be a proxy test for aflatoxin accumulation.     

磷对奶牛红细胞膜脂质的影响

对１５例临床上有明显低磷血症的奶牛血清磷、红细胞膜脂质成分进行了测定。结果表明，低磷血症奶牛血清磷、红细胞膜磷脂明显降低；红细胞膜胆固醇及膜胆固醇／磷脂摩尔比值（ｎｃｈ／ｎｐｌ）明显升高，与健康对照组牛相比差异极显著（Ｐ＜０．０１）；直线相关分析表明，血清磷与红细胞膜磷脂之间呈极显著正相关（ｒ＝０．９１７，ｙ＝１０．８５２ｘ＋３．１９６，Ｐ＜０．０１）；与红细胞膜胆固醇以及ｎｃｈ／ｎｐｌ之间分别呈显著负相关（ｒ＝－０．９４０，ｙ＝２．８５０ｘ－１．０７２，Ｐ＜０．０１；ｒ＝－０．９２０，ｙ＝１．９６８ｘ－１．４０１，Ｐ＜０．０１）。此外，红细胞膜微粘度与膜磷脂之间呈显著负相关（ｒ＝－０．９５４，ｙ＝１９．１２２ｘ－４．３８４，Ｐ＜０．０１），而与膜胆固醇以及ｎｃｈ／ｎｐｌ之间呈显著正相关（ｒ＝０．９８８，ｙ＝０．９２７ｘ＋０．９６４，Ｐ＜０．０１；ｒ＝０．９７８，ｙ＝－０．２９３ｘ＋１．１１３，Ｐ＜０．０１）。结果表明，磷是红细胞膜脂质成分改变的先决因素，而后者又是红细胞膜流动性及膜结构、功能发生变化并导致膜损伤的一个重要因素。     

应用FA及PPA-ELISA技术对猪瘟的检测

用免疫荧光抗体诊断技术及单克隆抗体纯化酶联免疫吸附试验诊断技术对疑为猪瘟病毒感染猪进行了检测 ,重点阐述了 2种方法的技术原理和操作过程。通过用免疫荧光抗体诊断技术检测了 5份病料 ,其中有 2份为阳性 ,阳性率为 40 % ;用单克隆抗体纯化酶联免疫吸附试验诊断技术检测了猪瘟血清抗体 ,在 40头母猪血清样品中 ,猪瘟弱毒抗体效价 OD值较高 ,有 1 0 0 %的保护率 ,但是发现母猪群中有 1 0 %隐性猪瘟感染 ,其强毒抗体效价 OD值大于 0 .5,体内带有猪瘟病毒。结果及过程表明此两种诊断方法检测快速、鉴别准确、分辨率高     

马传贫驴白细胞弱毒疫苗株跨膜蛋白主要免疫决定区基因的克隆与表达

从感染驴白细胞的马传贫驴白细胞弱毒疫苗株前病毒DNA中克隆了编码跨膜蛋白主要免疫决定区(TMIR)的基因，并在大肠杆菌中进行了表达。所表达的融合蛋白有一部分是可溶的，其氨基端带有6个组氨酸的标签，因此可以用固定化金属离子亲和层析法在非变性条件下进行纯化。在间接酶联免疫吸附试验(ELISA)和免疫印迹试验中，重组的TMIR蛋白可与马传贫阳性血清样品发生反应，而与健康马血清无任何反应。这表明该重组蛋白具有良好的抗原性和特异性，可用于马传贫弱毒疫苗株在体内外复制、接种马体内免疫应答及马传贫诊断的研究。     

甘肃滩羊血红蛋白(Hb)及红细胞蛋白质(Ep)多态性的研究

运用聚丙烯酰胺凝胶电泳技术(PAGE)对甘肃滩羊产区皋兰、景泰及靖远3县285只滩羊的血红蛋白(Hb)基因座、红细胞蛋白质-1(Ep-1)和红细胞蛋白质-2(Ep-2)基因座多态性进行了检测,结果发现甘肃滩羊Hb基因座存在3种基因型HbAA、HbAB、HbBB,它们受HbA和HbB两个共显性等位基因控制,其中HbB和HbBB在3个群体中均占优势;本研究未在Ep-1和Ep-2基因座上检测到多态性,它们均呈现单态。     

Neutrophil adherence,phagocytic-nitroblue tetrazolium reduction and chemiluminescence in canine whole blood

Methods for measuring neutrophil adherence, phagocytic-nitroblue tetrazolium (NBT) reducing activity and chemiluminescence were applied to canine whole blood as means for routine assessment of neutrophil functions. The phagocytic-NBT reduction test appeared to be useful for monitoring the NBT reducing activity of phagocytic cells associated with phagocytic functions. Ethylene diamine tetraacetic acid suppressed both the adherence and the phagocytic-NBT reducing activity of neutrophils. Increased phagocytic-NBT reduction and an enhanced chemiluminescence response were observed in dogs with neutrophilia. These methods provide a rapid and practical screening procedure for measuring selected phagocytic functions in canine whole blood.     

用酶联免疫吸附试验检测鸡白血病以及控制、根除鸡白血病措施的探讨

用酶联免疫吸附试验(ELISA),对2群鸡的鸡蛋清和1株鸡的马立克氏病疫苗进行检测,发现2群鸡的鸡蛋清中,鸡白血病病毒的阳性率分别是11％和29％,鸡马立克氏病冻干苗隐藏鸡白血病病毒群体特异性(gs)抗原的阳性率为100％。本文指出我国禽苗可能带有鸡的白血病病毒,分析讨论了鸡白血病病毒的垂直传递和水平传播的规律,提出在曾祖代和祖代鸡群中,采用ELISA试验,检测鸡蛋清,能减少以至根除鸡的白血病。     

Development of a semi-selective medium and an immunofluorescence colony-staining procedure for the detection of Clavibacter michiganensis subsp. sepedonicus in cattle manure slurry

Various compounds and basal media were tested for their suitability to create a semi-selective medium for isolation ofClavibacter michiganensis subsp.sepedonicus (Cms) from cattle manure slurry containing c. 10⁸ colony forming units (cfu) per ml.Plating efficiency of Cms in yeast glucose mineral medium (YGM) was 104% compared with yeast peptone glucose medium. Nalidixic acid, polymyxin B sulphate and the experimental disinfectant S-0208 inhibited colony growth of cattle slurry bacteria as compared with Cms in YGM. The optimal concentration of these inhibitors in combination was determined by modified agar diffusion tests and by pour plating in 24-well tissue culture plates. The semi-selective medium YGMI consisted of YGM supplemented with nalidixic acid (2 mg/l), polymyxin B sulphate (30 mg/l) and S-0208 (125 mg/l). Plating efficiency varied for Cms between 50.9 and 69.6%, for cattle slurry bacteria between 1.8 and 2.5% and for saprophytes from potato heel end extracts between 11.5 and 27.4%.Differentiation of Cms colonies from other colonies was based on their small and bluish colony morphology in pour plates and on immunofluorescence colony-staining (IFC). IFC of a pure culture of micro colonies of Cms in YGM was possible after one day incubation (colonies c. 5 cells). Green background fluorescence in the agar gels was prevented by addition of Tween 20 (0.1%) to the washing buffer and the use of 1% agar gels. IFC of macro colonies of Cms in YGMI, visible with 4x objective magnification, was possible after 4 days. The detection level of the target organism in artificially inoculated cattle slurry in YGMI based on colony morphology varied between 1.4×10³ and 2.3×10⁴ cfu per ml of cattle slurry. Miniaturized plating combined with IFC, using wells in tissue culture plates (=16 mm), proved suitable for detection, but was c. 30 times les sensitive. The recovery of Cms was negatively correlated with the number of saprophytic colonies in the agar plates (R ²=0.74).     

Biochemical properties of the pathogenesis-related proteins from tobacco

This review describes the discovery and identification of the pathogenesis-related proteins (PRs) from tobacco. In crude leaf extracts the PRs are distinguished from the proteins in uninfected plants by their solubility at pH 3, resistance to a range of proteases, and mobility in polyacrylamide gels upon electrophoresis (PAGE) in non-denaturing conditions. PAGE has been used as a qualitative and semi-quantitative assay for PRs, and their migration in gels made from different acrylamide concentrations has been used to identify charge and size isomers and electrophoretically identical PRs in different tobacco cultivars. The subunit composition and molecular weight (mol. wt) of the four PRs identified first in Xanthi-nc were determined by SDS-PAGE; staining the gels has shown that these same four proteins in Samsun NN did not contain carbohydrate, lipid or nucleic acid, nor were they isozymic forms of twenty five enzymes known to increase in activity following infection with TMV. Evidence suggests that most of the PRs in Xanthi-nc and Samsun NN are extracellular.The purification of several PRs from Xanthi-nc, Samsun NN and other tobaccos is described, as well as their mol. wt, subunit and amino acid composition. PRs 1a, b and c consist of a single polypeptide and have similar mol. wt and amino acid compositions. Antisera prepared against purified Xanthi-nc b₁ protein have been used to determine serological relationships between PRs and form the basis of a very sensitive quantitative assay using ELISA. The regulation of synthesis of some PRs has been shown to involve translational control.