Eisenia fetida中蚯蚓纤溶酶P-Ⅲ组分序列多态性

为了探讨蚯蚓纤溶酶P—Ⅲ组分序列多态性及其与蚓种关系。从Eisenia fetida中提取总RNA,利用P-Ⅲ组分序列两端的保守性设计引物,通过RT—PCR获得P—Ⅲ组分基因,并构建该组分质粒（pUC18）文库,经PCR筛选后进行序列测定及分析。在原已获得的Efp-Ⅲ-1和Efp-Ⅲ-3的基础上,利用中间引物从文库中筛选到Efp-Ⅲ-2基因。序列分析表明,该基因编码序列为720bp,编码239个氨基酸,与GenBank报道的点Efp-Ⅲ-2五条序列整体相似性高达99．02％。通过序列比对,发现Efb-Ⅲ-1、2和3具有完全相同的结构域,差异位点零星分布在序列中间;Lumbricus rubellus的六条序列同样具有Eisenia fetida这个特点,并无明显差异。序列比对证明它们是蚯蚓体内P-Ⅲ组分基因多态性的表现,而且在Lumbricidae科中与蚓种无关。     

Toxicokinetics and Biotransformation of As(III) and As(V) in Eisenia fetida

Earthworms, Eisenia fetida, were exposed to soils spiked with As(III) and As(V), to understand the response of earthworms to As in terms of both toxicity and accumulation using toxicokinetics, and to explain As metabolism and bioavailability. As(III) showed higher toxicity than As(V), in all toxicity endpoints of burrowing time, survival, growth, and cocoon production. Bioconcentration occurred at both As(III) and As(V) treatments during 28-d exposure. Worms did not show the elimination of As for consecutive 28 d of exposure to clean soils. Biotransformation of As was characterized using HPLC-ICP-MS and XANES, showing the reduction of As in worms regardless of the As species to which worms were exposed. Metabolism of As in worms that formed As-thiol complex is thought to limit the excretion of As, and thus induce bioconcentration in worms. Uptake rates by one-compartment model indicated that pore water was the bioaccessible pool of As, and directly controlled the uptake of As by worms. The study suggests that higher uptake rate and bioaccumulation of As(III) than of As(V) are among the factors that make As(III) more toxic than As(V).     

Bioremediation of Agricultural Wastes through Vermicomposting

The post-harvest residues of some local crops, e.g., wheat (Triticum aestivum), millets (Penniseum typhoides and Sorghum vulgare), and a pulse (Vigna radiata) were subjected to recycle through vermicomposting. The crop residues were amended with animal dung, and three types of vermibeds were prepared: (i) millet straw (S. vulgare + P. typhoides in equal quantity) + sheep manure (1: 2 ratio) (MS); (ii) pulse bran (V. radiata) + wheat straw (T. aestivum) + cow dung (1: 1: 2 ratio) (PWC); and (iii) mixed crop residues (mixing of all types crop residues, used in this study) + cow dung in 1:1 ratio (MCR + CD). The fourth treatment was cattle shed manure (CSM). Vermicomposting resulted in a significant increase in total N (97.3% to 155%), available P (67.5% to 123.5%), exchangeable K (38.3% to 112.9%), and exchangeable Ca (23.3% to 53.2%), and decrease in organic C content (20.4% to 29.0%) in the different vermibeds. The earthworm showed the higher biomass gain, growth rate (mg wt. worm^?1 day^?1) and cocoon numbers in the CSM vermibed. The quality of crop residues was directly related to the rate of organic matter mineralization during the vermicomposting. This study suggests that agriculture wastes could be converted into some value-added products, e.g., vermicompost and worm biomass through vermicomposting technology. The higher concentrations of plant nutrients in end products indicate a potential for using agriculture wastes in sustainable crop production.     

Multi-isomorphous replacement phasing of the earthworm fibrinolytic enzyme component A from Eisenia fetida

Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida, a protein functioning not only as a direct fibrinolytic enzyme, but also as a plasminogen activator, has been crystallized in P2₁2₁2₁ space group with 3 protein molecules per asymmetric unit. Four heavy atom derivatives were prepared using a mother liquor containing 1.4 mol · L^-1 Li₂SO₄ and 0.1 mol · L^-1 MOPS buffer (pH7.2) and used to solve the protein’s diffraction phase. The heavy atom binding sites in the derivative crystals were determined using difference Patterson and difference Fourier methods and were refined in combination to yield the initial protein’s structure phase at 0.25 nm resolution. The non-crystallographic symmetry relationship of the three independent protein molecules in the asymmetric unit was determined using the correlative heavy atom sites and used for the averaging of the initial electron density. As a result, the electron density was significantly improved, providing a solid foundation for subsequent structure determination.     

蚯蚓纤溶酶基因(Efp-Ⅰ)在大肠杆菌中的克隆、表达及活性分析

从GenBank查询到的蚯蚓纤溶酶(earthworm fibrinolyti cenzyme,EFE)基因序列中,只有AY438624翻译的蛋白质序列与天然EfP-Ⅰ在N-端具有较高的相似性。根据该基因的5′与3′序列设计引物,通过RT-PCR从赤子爱胜蚓(Eisenia fetida)获得一个完整的基因(GenBank,DQ418454)。序列分析证明,由该基因编码蛋白质的N-末端与天然EfP-Ⅰ的N-末端的氨基酸顺序完全相同。ScanProsite prediction programs分析显示,该基因与AY438624相似性极高,二者均属于胰蛋白酶家族;不同的是,该基因编码蛋白质的序列中含有N-糖苷键的结构域,所以DQ418454是EfP-Ⅰ中的一个新基因。在此基础上,构建了该基因的原核表达载体pMAL-c2X-Efp-Ⅰ,并进行了转化、诱导和表达。Western blotting证明,表达产物同时具有MBP和EfP-Ⅰ的抗原特异性,是MBP和EfP-Ⅰ的融合蛋白(MBP-EfP-Ⅰ)。经亲和层析分离纯化的MBP-EfP-Ⅰ,在酪蛋白平板和纤维蛋白平板上表现出明显的纤溶酶活性。     

Multi-isomorphous replacement phasing of the earthworm fibrinolytic enzyme component A from Eisenia fetida

Thrombosis is one of the most widely occurring diseases in modern life, which often causes disability and death. Fibrinolytic enzymes degrade fibrin, the major protein component of blood clots, and eventually lead to thrombolysis. Medications using fibrinolytic enzymes are the most effective methods used in the treatment of thrombosis. A variety of fibrinolytic enzymes, such as tPA, uPA, and streptokinase, have been extensively studied and used as thrombolytic agents in clinic. However, thes…     

Protective effect of composite earthworm powder against diabetic complications via increased fibrinolytic function and improvement of lipid metabolism in ZDF rats

Thrombosis is the leading cause of mortality globally. It is not only a complication but also a risk factor for progression of diabetes. However, alternative oral therapies and prophylaxis with less adverse effect for thrombosis have not been well studied. In this study, composite powder containing earthworm (CEP) was used and its fibrinolytic activity was measured. CEP was found to have a high urokinase-type plasminogen activator like activity in an in vitro assay. It also had significantly shortened euglobulin clot lysis time (ECLT) at 4 and 24 h after ingestion in Sprague Dawley rats. Zucker Diabetic Fatty rats were used to assess the effect of CEP on diabetes and diabetic nephropathy. After 10 weeks of feeding, CEP significantly shortened ECLT and attenuated HbA1c, hepatic lipid accumulation, and urinary albumin excretion and improved glomerular mesangial matrix score. Therefore, CEP may have beneficial effects on diabetes and diabetic nephropathy.     

视黄酸对赤子爱胜蚓再生头尾轴形成的影响

为了探讨视黄酸对蚯蚓再生的影响,用视黄酸处理了从不同部位剪切的蚯蚓体段．观察其存活率、重量和再生长度的变化。结果表明,有头无尾的体段存活率受视黄酸影响较小,而无头有尾的处理受视黄酸影响较大;视黄酸处理后30d,各处理再生长度和存活率均小于对照;视黄酸对蚯蚓再生有明显影响,能延迟和干扰再生,影响头部的形成。视黄酸影响蚯蚓再生的作用方式可能是通过干扰前后体轴的形成,从而影响蚯蚓再生图式形成。     

赤子爱胜蚓再生过程中肌细胞超微结构变化的研究

将赤子爱胜蚓在60／61体节处剪切,用透射电镜观察肌肉再生过程中的形态变化。结果表明：剪切后伤口处的受损细胞呈降解状态并被迁移至伤口面的吞噬细胞所吞噬;伤口处的其他未受损细胞去分化,失去特化细胞的特征,并且增殖形成圆锥状的芽基;芽基细胞再次进行分化,成肌细胞合成肌丝,最终生成新的肌肉组织。     

Reconfirmation of antimicrobial activity in the coelomic fluid of the earthwormEisenia fetida andrei by colorimetric assay

A novel tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) was used in the assessment of antimicrobial activity in earthworm in the presence of phenazine methosulphate (PMS) as an electron coupling reagent. This activity was purified from the coelomic fluid of the earthworm (ECF),Eisenia fetida andrei (Oligochaeta, Lumbricidae, annelids) using a series of column chromatography techniques and was tested against three Gram-negative strains ofEscherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and three Gram-positive strains ofStaphylococcus aureus, Bacillus megaterium, Arthrobacter sp., respectively. Only the pigment-free eluate of coelomic fluid of the earthworm (ECFPE) showed activity againstB. megaterium amongst three isolated active fractions. The anion (DEAE-52) exchange effluent of the ECFPE was reported to have the strongest activity againstP. aeruginosa amongst the three active fractions. The 20% acetonitrile eluate (AE) by Sep-Pak C₁₈ cartridge was also tested and showed fair resistance againstE. coli, P. aeruginosa andArthrobacter sp., respectively.     

9种农药对家蚕和蚯蚓的急性毒性

【背景】当前农药品种及其使用量日益增多,测试农药对环境生物的急性毒性成为农药环境安全监测的重要途径。【方法】采用食下毒叶法和药土法分别测定了甲氨基阿维菌素苯甲酸盐、阿维菌素、氰氟虫腙、螺螨酯、螺虫乙脂、嘧菌酯、苯醚甲环唑、戊唑醇、2,4-D二甲胺盐等9种农药对家蚕和蚯蚓的急性毒性,并根据其毒性等级划分标准进行分级,评价其对环境的安全性。【结果】甲氨基阿维菌素苯甲酸盐、阿维菌素、氰氟虫腙、螺螨酯、螺虫乙脂、嘧菌酯、苯醚甲环唑、戊唑醇和2,4-D二甲胺盐对家蚕的LC50(96 h)分别为2.05×10-3、8.59×10-4、2.79、250.48、11.52、272.18、2.50、1.93×10-2和534.47 a.i.mg·L-1,对蚯蚓的LC50(14 d)分别为11.05、6.29、100、100、100、100、99.13、115.31和100 a.i.mg·kg-1干土。其中,甲氨基阿维菌素苯甲酸盐、阿维菌素和戊唑醇对家蚕\"剧毒\",氰氟虫腙、螺虫乙脂和苯醚甲环唑对家蚕\"高毒\",其余农药对家蚕均为\"低毒\";阿维菌素对蚯蚓为\"中毒\",其余农药对蚯蚓均为\"低毒\"。【结论与意义】9种农药对家蚕和蚯蚓的急性毒性存在差异,为农药的合理使用和环境保护提供了依据。     

蚯蚓纤溶酶基因的克隆与序列分析

目的:获取蚯蚓纤溶酶(EFE)的基因,并对其进行序列分析。方法:从赤子爱胜蚓中提取总RNA,然后利用RT-PCR技术扩增EFE的基因,并将其插入pUCm-T载体,经PCR和酶切鉴定后进行序列测定及分析。结果:DNA序列分析表明,所克隆的EFE基因全长为738bp,其中编码区段为735bp,共编码245个氨基酸残基,成熟肽为238个氨基酸残基。与GenBank中已报道的粉正蚓的EFE序列F-Ⅲ-2的同源性最高,两者在核苷酸序列上有2处不同,即第137位(T→C)和第632位(G→T),密码子也因而分别由GTC、GGT变为GCC、GTT,导致第46位、211位的氨基酸残基分别由缬氨酸、甘氨酸变为丙氨酸和缬氨酸。结论:从赤子爱胜蚓中成功克隆了1条EFE基因F245。序列测定及同源性分析表明,蚯蚓纤溶酶F245与已报道的多条EFE基因序列具有高度同源性,且具备完整的编码区。该序列的克隆为用基因工程的方法生产单一成分的高酶活性EFE奠定了基础。     

蚓激酶基因的克隆及在毕赤酵母中的表达

以赤子爱胜蚓(Eisenia fetida)体内的总RNA为模板,通过RT-PCR方法扩增含自身信号肽的蚓激酶基因F238,将其克隆到pUCm-T载体上,并进行测序。GenBank登录号为:DQ202401。测序结果表明基因全长为738bp,共编码245个氨基酸,包括7个氨基酸的信号肽序列和238个氨基酸的成熟肽序列。与粉正蚓(Lumbricus rubellus)F-III-2相比,核苷酸与氨基酸序列的同源性均为99%,仅存在2个碱基的差异,导致2个氨基酸的突变。通过生物信息学方法对蛋白质的理化及结构特性进行分析预测,F238的等电点为4.61,含有11个半胱氨酸,形成3个二硫键。蛋白质分子主要由β折叠组成,具有丝氨酸活性中心,属丝氨酸蛋白酶超家族胰蛋白酶类。以重组质粒pUCm-T-F238为模板,通过PCR方法扩增去信号肽的蚓激酶基因F238-m,构建毕赤酵母(Pichia pastoris)表达载体pPIC9-F238-m,将其线性化后用电穿孔法导入酵母宿主菌GS115中。在MM和MD平板上筛选表型,经甲醇诱导后,SDS-PAGE分析显示表达产物的分子量为28kDa左右,纤维平板法测定活力最高可达100U/mL。