检测大肠杆菌(Escherichia coli) O157:H7免疫磁珠的制备、保存与应用

【背景】大肠杆菌(Escherichia coli) O157:H7是导致肠出血性大肠杆菌食源性疾病暴发的主要血清型,免疫磁珠(Immunomagnetic beads,IMBS)在E. coli O157的检测中发挥着重要作用,而免疫磁珠的稳定性、特异性、广谱性等性能指标关系着在实际应用中的使用效果。【目的】制备高效、稳定且具有广谱性的免疫磁珠,联合分子检测技术如环介导恒温扩增 (Loop-mediated isothermal amplification,LAMP)技术、PCR等,提高目标菌的检出率。【方法】采用新型的磁珠活化剂MIX&GO制备E. coli O157免疫磁珠,并进行广谱性以及特异性检测;针对6种试剂牛血清白蛋白(Bovine serum albumin,BSA)、酪蛋白(Casein)、海藻糖(Trehalose)、聚乙烯吡咯烷酮(Polyvinyl pyrrolidone,PVP)、抗坏血酸(Vitamin C)和防腐剂ProClin 300,利用正交试验L18(37)优化免疫磁珠保存液组分;采用IMBS-LAMP、IMBS-PCR、IMBS-生化、菌液-LAMP、菌液-PCR、显色平板-生化鉴定6种方式对20份生猪肉样品进行检测。【结果】利用MIX&GO活化剂制备的免疫磁珠捕获率最高达到81.5%±1.3%;免疫磁珠保存液最优配方为：牛血清白蛋白15.0 g/L,酪蛋白10.0 g/L,海藻糖10.0 g/L,PVP 2.0 g/L,抗坏血酸5.0 g/L,ProClin 300 2.5 g/L,保存6个月后免疫磁珠捕获率为75.5%;在20份生猪肉样品的检测中,自制磁珠和商品化磁珠与LAMP联用均检出9例阳性样品;IMBS-LAMP在6种检测方式中具有最高的检测灵敏度,但检出的样品会因磁珠抗体的差异而有所不同。【结论】与商品化磁珠相比,实验制备的免疫磁珠具有良好的特异性和广谱性,免疫磁珠-LAMP联用提高了目标菌的检出率,是一种高灵敏度、具有应用前景的检测方法。     

环境无机汞对泥鳅视网膜S100表达影响

用含低浓度Hg2 的水饲养泥鳅(Misgurnus anguillicaudatus),研究Hg2 污染对视网膜中S100免疫反应阳性结构数量及分布的影响。用免疫组织化学ABC法标记S100免疫阳性(S100-IR)细胞,显微镜下观察S100免疫反应阳性细胞并计数。结果显示,处理组S100首先在神经节细胞层(GCL)、神经纤维层(NFL)表达,然后逐渐在其他各层表达。与对照组相比,处理组S100免疫反应阳性细胞数显著增多,且该变化与环境中无机汞处理时间成正相关;阳性细胞胞体明显膨胀,突起稠密粗大,S100阳性反应强。研究提示,Hg2 的神经毒害作用导致泥鳅视网膜中S100-IR细胞数的增多。推测Hg2 污染可能对视网膜有伤害性影响,而神经胶质细胞在视网膜修复过程中起了重要作用。     

中国不同地域恒河猴MHC-Ⅰ型部分等位基因的调查

采用序列特异性引物-聚合酶链式反应(PCR-SSP)分型方法对在华南灵长类动物研究中心繁殖的247只中国恒河猴(Macaca mulatta)(其中30只来源于广西、34只来源于海南,183只来源于川西、安徽等内陆地区杂交群)的Mamu-A01、A02、A08、B01和NA7等5个MHC-Ⅰ型分子位点进行检测。结果显示,A01、A02发现于海南群和杂交群,阳性率均小于8.8%;A08发现于杂交群中,阳性率小于3.8%;B01发现于广西群和杂交群,阳性率均大于17.48%;NA7在3个猴群中均有发现,阳性率均大于32.4%。中国不同地域恒河猴群体携带的5个MHC-Ⅰ型等位基因的频率存在明显差异,通过与印度的恒河猴比较,中国恒河猴与印度恒河猴携带的MHC-Ⅰ等位基因也存在显著的差异。     

Enzymatic assembly of epothilones: the EpoC subunit and reconstitution of the EpoA-ACP/B/C polyketide and nonribosomal peptide interfaces

The biosynthesis of epothilones, a family of hybrid polyketide (PK)/nonribosomal peptide (NRP) antitumor agents, provides an ideal system to study a hybrid PK/NRP natural product with significant biomedical value. Here the third enzyme involved in epothilone production, the five domain 195 kDa polyketide synthase (PKS) EpoC protein, has been expressed and purified from Escherichia coli. EpoC was combined with the first two enzymes of the epothilone biosynthesis pathway, the acyl carrier protein (ACP) domain of EpoA and EpoB, to reconstitute the early steps in epothilone biosynthesis. The acyltransferase (AT) domain of EpoC transfers the methylmalonyl moiety from methylmalonyl-CoA to the holo HS-acyl carrier protein (ACP) in an autoacylation reaction. The ketosynthase (KS) domain of EpoC decarboxylates the methylmalonyl-S-EpoC acyl enzyme to generate the carbon nucleophile that reacts with methylthiazolylcarboxyl-S-EpoB. The resulting condensation product can be reduced in the presence of NADPH by the ketoreductase (KR) domain of EpoC and then dehydrated by the dehydratase (DH) domain to produce the methylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediate that serves as the acyl donor for subsequent elongation of the epothilone chain. The acetyl-CoA donor can be replaced with propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA and the acyl chains accepted by both EpoB and EpoC subunits to produce ethyl-, isopropyl-, and phenylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediates, suggesting that future combinatorial biosynthetic variations in epothilone assembly may be feasible. These results demonstrate in vitro reconstitution of both the PKS/NRPS interface (EpoA-ACP/B) and the NRPS/PKS interface (EpoB/C) in the assembly line for this antitumor natural product.     

Pluripotent stem cells from the adult mouse inner ear

In mammals, the permanence of acquired hearing loss is mostly due to the incapacity of the cochlea to replace lost mechanoreceptor cells, or hair cells. In contrast, damaged vestibular organs can generate new hair cells, albeit in limited numbers. Here we show that the adult utricular sensory epithelium contains cells that display the characteristic features of stem cells. These inner ear stem cells have the capacity for self-renewal, and form spheres that express marker genes of the developing inner ear and the nervous system. Inner ear stem cells are pluripotent and can give rise to a variety of cell types in vitro and in vivo, including cells representative of ectodermal, endodermal and mesodermal lineages. Our observation that these stem cells are capable of differentiating into hair cell-like cells implies a possible use of such cells for the replacement of lost inner-ear sensory cells.     

噬菌体DNA的快速抽提

介绍一种噬菌体DNA的快速抽提方法.用聚乙二醇沉淀噬菌体颗粒,然后经DEAE纤维素纯化处理和酚抽提.与传统的噬菌体DNA纯化方法相比,改进后的方法方便、快速、经济,可获得高纯度的噬菌体DNA.     

Immunolcocalization of actin in intact and DNA—and histone—depleted nuclei and chromosomes of allium cepa

The presence of actin in eukaryotic nuclei and chromosomes,and especially in higher plant nuclei and chromosomes,has not been well established.We detected actin in meristematic cells of Allium cepa with indirect immunofluorescence technique and observed bright fluorescence in the intact nuclei and chromosomes,indicating that actin is present in the nuclei and chromosomes of the higher plant.We labeld sections of the meristematic cells of A.cepa with immunogold technique,gold parti cles were concentrated in condensed chromatin and nucleoli,confirming the results of the immunofluoresence observations.We traeated the nuclei and chromosomes of A.cepa with DNase I and 2M NaCl and obtained DNA-and histone-depleted nuclei and chromosomes.Indirect immunofluorescence tests showed that the DNA-and histonedepleted nuclei and chromosomes reacted positively with the anti-actin antibodies.These results demonstrate that the anti-actin antibodies.These results demonstrate that actin exists not only in intact nuclei and chromosomes,but also in DNA-and histone-depleted nuclei and chrmosomes of the plant.In addition,our immuno-fluorescence tests indicate that tropomyosin is present in the nuclei and chromosomes of A.cepa.     

水稻(Oryza sativa)原生质体经钝化后诱导融合再生可育体细胞杂种

本研究在初步实现水稻原生质体培养的程序化后,选用普通栽培稻P339和特种稻苏御糯选的原生质体为融合亲本,利用碘乙酸(IA)和罗丹明-6G(R-6G)这两个代谢互补抑制剂钝化处理亲本原生质体,确定了合适的抑制条件。P339用0.25mmol/L IA,苏御糯选用50μg/ml R-6G分别经30min钝化处理,通过PEG和高Ca~(2 )、高pH法诱导融合,异源融合体具有代谢互补效应,经培养得到愈伤组织17块,并进一步分化获得不同形态的再生植株12棵。移栽存活的再生植株成熟后可育,通过对这些植株的形态以及酯酶和过氧化物酶同工酶电泳的分析表明是融合后的体细胞杂种植株。