苏云金芽孢杆菌cry1Aα核心毒素基因转化烟草及其杀虫活性

将苏云金芽孢杆菌cry1Aα的C端编码区截短，获得编码N端609个氨基酸约1.8kb的核心毒素基因，将其构建到植物表达载体转化烟草，研究直接表达的活性ICP核心毒素被昆虫取食后的杀虫效果。T1代种子发芽的抗生素抗性分离和PCR检测证实cry1Aα核心毒素基因整合到了烟草基因组，Western杂交检测表明转化烟草能够正常表达大小为75．6kD的核心毒素蛋白。生物测定结果表明，转化烟草对初孵棉铃虫平均有高于60％的致死率，对初孵甜菜夜蛾平均达到70％的致死率，最高致死率均可达到90％以上，并对昆虫的生长发育有明显的抑制作用。     

苏云金芽孢杆菌cry1Aa核心毒素基因转化烟草及其杀虫活性

将苏云金芽孢杆菌cry1Aa的C端编码区截短,获得编码N端609个氨基酸约1.8kb的核心毒素基因,将其构建到植物表达载体转化烟草,研究直接表达的活性ICP核心毒素被昆虫取食后的杀虫效果.T1代种子发芽的抗生素抗性分离和PCR检测证实cry1Aa核心毒素基因整合到了烟草基因组,Western 杂交检测表明转化烟草能够正常表达大小为75.6kD的核心毒素蛋白.生物测定结果表明,转化烟草对初孵棉铃虫平均有高于60%的致死率,对初孵甜菜夜蛾平均达到70%的致死率,最高致死率均可达到90%以上,并对昆虫的生长发育有明显的抑制作用.     

分泌抗Bt Cry1Ac蛋白单克隆抗体杂交瘤细胞株的建立

从苏云金芽孢杆菌HD-73中提取Bt Cry1Ac蛋白,电镜观察Bt Cry1Ac蛋白为标准的菱形。用SP2／0-Ag14骨髓瘤细胞与经该Bt Cry1Ac蛋白免疫的BALB／c小鼠的脾细胞融合,经3次克隆化,筛出两株稳定分泌Bt Cry1Ac单克隆的杂交瘤细胞株1C3、2F3。两株细胞均具有抗Bt Cry1Ac特异性,与Bt Cry1Ab和Bt Cry2A无明显的交叉反应,经亚型鉴定均为IgG1。腹水滴度均为1：1024000。     

两段杨树叶绿体DNA片段的克隆及叶绿体定点转化载体的构建

利用PCR方法,从毛白杨（Populus tomentosa C.）叶绿体基因组中克隆了1．7kb和1．6kb的相邻DNA片段,对其进行序列分析表明,扩增片段分别具有1766个和1601个核苷酸,前者包括3′rps12,rps7基因的编码区及其边界序列,后者包含ndhB基因的第一外显子和内含子。本文还构建了杨树这两个片段的限制酶切图谱,并以这两个相邻片段为同源重组片段,分别将绿色荧光蛋白（GFP）基因和苏云金芽孢杆菌杀虫蛋白（Bt）基因,及其原核表达框插入其间,构建了杨树叶绿体定点转化载体pPZG和pPZB。这两个特异性载体将定位整合到杨树叶绿体基因组中反向重复区的rps7和ndhB基因的间隔区,并高效表达GFP和Bt基因。迄今为止,本文所报道的内容在国内、外尚属首次。     

甜蛋白Monellin在毕赤酵母中的分泌表达

在西非热带植物Dioscoreophyllum cumminsii中存在着一种由两条多肽构成的甜度极高的蛋白Monellin。本研究根据已知的Monellin晶体衍射分析结果和毕赤酵母(Pichia pastoris)基因偏爱密码子设计并合成了连接两条多肽的重组基因，插入到毕赤酵母分泌表达质粒pGAPZαA中。扩增后，电击穿孔转化毕赤酵母GS115，筛选高产菌株放大培养。以5升罐发酵培养，表达量为150mg／L。经纯化，可以获得大于130mg／L的具有高甜度的活性蛋白。     

C端带多聚组氨酸标签的重组小鼠基质细胞衍生因子-1α原核系统的表达及分离纯化

在构建SDF-1原核表达系统的基础上，探索对其活性表达产物分离纯化的技术路线。将从小鼠骨髓组织克隆出的SDF-1a成熟肽基因插入原核表达质粒pET101／D-FOFO，用以转化大肠杆菌B1221Star^TM菌株，经SDS-PAGE进行诱导表达。SDS-PAGE和Western Blot检测证实：其表达产物在约11．6kD处有明显条带显示，其大小与预测的重组SDF-1a／His分子量一致。采用Ni^2 -Resin固相亲和层析法对由该系统表达的C端带6Xhis标签的重组SDF-1a／His进行分离纯化，纯度达92％。体外活性检测结果表明：重组小鼠SDF1a／His对T细胞有明显趋化和促生存作用；能有效诱导Jurkat细胞ERK磷酸化。     

高效降解苯胺的菌株AD9的分离、生理生化特性分析和分子鉴定

从活性污泥中分离到一株能以苯胺为唯一碳源、氮源生长的苯胺降解菌株AD9,该菌株最适生长的苯胺浓度为1000mg／L,降解效率可达90％,对苯胺耐受程度高达4500mg/L,降解苯胺的最适pH值为7．0,最适温度为30℃。以上实验结果表明,AD9具有降解苯胺速率快、耐受苯胺浓度高的特性。该菌具有氨苄青霉素、卡那霉素和利福平抗性,无氯霉素、四环素抗性,其16SrDNA序列与多株Delftia sp．菌有很高的同源性,其G＋C含量为66．8mol％,非常接近于标准菌株D．tsuruhatensis T7（66．2mol％）。另外该菌和17的DNA-DNA杂交率为83．8％。结合AD9的表型鉴定将该菌株归属为D．tsuruhatensisas。这是D．tsuruhatensis菌种苯胺降解细菌菌株的首次报道。     

中国明对虾凝血栓蛋白基因全长cDNA的克隆及其表达特征

获得了中国明对虾凝血栓蛋白(TSP)基因全长cDNA,该基因由2868个碱基组成,具有一个2763bp的开放阅读框,编码920个氨基酸,其中包含21个氨基酸组成的信号肽.中国明对虾TSP由四类不同的结构域组成,从N端开始顺序为几丁质结合结构域,EGF-like结构域,Type Ⅲ repeat和TSP carboxyl-terminal domain(TSP-C结构域).结构域分析发现,中国明对虾的N端不具有TSP N-terminal domain(TSPN结构域),但有一个几丁质结合结构域,其C端的Type Ⅲ和TSP-C结构域非常保守.该基因在弧菌感染后的对虾淋巴器官和肝胰脏中的表达量显著增加,并具有不同的时空表达趋势,提示中国明对虾TSP基因在免疫反应中具有重要作用.     

水稻矮缩病毒非结构蛋白Pns11的核酸结合活性分析

水稻矮缩病毒的非结构蛋白Pns11是一个序列非特异性的核酸结合蛋白,其N端有类似锌指蛋白的结构,C端富含碱性氨基酸,为了研究Pns11的核酸结合活性,构建了两个位于可能的锌指结构内的点突变以及四个分别删除部分碱性区氨基酸片段的缺失突变,经测序验证后分别克隆到大肠杆菌表达载体pBV221中,温度诱导表达,用提取包涵体的方法初步纯化突变体蛋白,Western印迹检测这些突变体和Pns11的抗体均有特异反应,Csouthwestern和Northwestern印迹的方法证明六个突变体都保持了核酸结合活性,结果表明N端的类似锌指蛋白的结构不是结合核酸的活 位点,部分缺失C端的碱性氨基酸也不影响其结合。     

油菜叶绿体定点转化载体的构建及其杀虫性

首先从油菜叶绿体基因组克隆得到包含ｒｐｓ７基因在内的１．０ｋｂＤＮＡ片 包含ｎｄｈＢ基因在内的２．４ｋｂＤＮＡ片段,同时从苏云芽孢杆菌质粒上克隆得到一个全长３．５ｋｂ的ＢＴ杀虫蛋白基因ｃｒｙ１Ａａ。然后以ｒｐｓ７和ｎｄｈＢ基因作为同源重组片段,成功构建了包含Ｂｔ杀虫蛋白和ａａｄＡ抗壮观霉素基因的油菜叶绿体定点转化载体,并对克隆菌体总蛋白进行了生物杀虫试验。结果表明,Ｂｔ杀虫蛋白基因能够得到表达,并     

苏云金芽孢杆菌cry3Aa启动子的克隆和营养期表达载体的构建

苏云金芽孢杆菌（Bacillus thuringiensis,Bt)的绝大多数杀虫晶体蛋白（insecticidal crystal proteins,ICPs)基因是在芽孢形成期（sporulation phase)表达的,而只有少数基因如cry3Aa是在营养期表达的。在本研究中,根据已知的cry3Aa基因启动子序列设计了一对引物（Ep-5s和Ep-3n),利用这对引物从拟步虫甲亚种（Bt subsp.tenebrions)中扩增出一个与预期大小（1．1kb)一致的DNA片段。序列测定及分析结果表明,这个DNA片段含cry3Aa启动子全序列,包括上游AT富含区、两个启动子区、两个SD序列及两组反向重复序列。经过一系列的克隆之后将这个片段克隆到穿梭载体pHT3101上,最后构建成一个Bt的营养期表达载体pHPT。     

Comparison of the Mechanical Behaviour of Standard and Auxetic Foams by X‐ray Computed Tomography and Digital Volume Correlation

The tensile behaviour of standard and auxetic polyurethane foams are contrasted by digital volume correlation of 3D images collected by in situ X‐ray computed tomography (CT). It was found that subset sizes of 32 and 64 voxels for the auxetic and standard foams were optimal for strain resolutions in the order of 0.1%. For the standard foam, good uniformity of strain was observed at low strains giving a tangent Poisson's ratio of 0.5. Some heterogeneity of strain was observed at higher strains, which may be related to the fixtures. The behaviour of the auxetic foam was totally different, with strain being spatially heterogeneous with transverse strains both positive and negative but giving a negative Poisson's ratio on average. This suggests that the unfolding tendency of some groups of cells was higher than others because of the complex frozen starting microstructure. Further different methods of deriving Poisson's ratio gave different results. Besides revealing interesting microstuctural mechanisms of transverse straining, the study also shows digital volume correlation of tomography sequences to be the perfect tool to study complex mechanical behaviour of cellular materials.     

Bioluminescence detection system of mutagen using firefly luciferase genes introduced in Escherichia coli lysogenic strain.

A rapid and convenient microbial sensing system for mutagens was developed based upon the induction of prophage from Escherichia coli lysogenic strain and bioluminescence. The system consisted of lysogenic E. coli encoding firefly luciferase genes and a photodetection system. Measurement of mutagen mitomycin C was achieved by measuring the luminescence intensity emitted from E. coli lysogenic strain for the recombinant phage in the presence of luminescence substrates. Approximately 1 h after addition of mitomycin C, the luminescence began to be observed, and 3 h after, it attained a level of 2 times greater than that of 1 h. Irradiation with ultraviolet light also produced light based on induction of phage from the E. coli lysogenic strain for the recombinant phage. On the other hand, when nonmutagenic toxic compounds like sodium azide were added to the reaction medium, luminescence was not observed. Mitomycin C could be detected within 1 h with this sensing system, at concentrations down to 10(2) ng/assay.     

Textile dye degradation by bacterial consortium and subsequent toxicological analysis of dye and dye metabolites using cytotoxicity, genotoxicity and oxidative stress studies

The present study aims to evaluate Red HE3B degrading potential of developed microbial consortium SDS using two bacterial cultures viz. Providencia sp. SDS (PS) and Pseudomonas aeuroginosa strain BCH (PA) originally isolated from dye contaminated soil. Consortium was found to be much faster for decolorization and degradation of Red HE3B compared to the individual bacterial strain. The intensive metabolic activity of these strains led to 100% decolorization of Red HE3B (50 mg l(-1)) with in 1h. Significant induction of various dye decolorizing enzymes viz. veratryl alcohol oxidase, laccase, azoreductase and DCIP reductase compared to control, point out towards their involvement in overall decolorization and degradation process. Analytical studies like HPLC, FTIR and GC-MS were used to scrutinize the biodegradation process. Toxicological studies before and after microbial treatment was studied with respect to cytotoxicity, genotoxicity, oxidative stress, antioxidant enzyme status, protein oxidation and lipid peroxidation analysis using root cells of Allium cepa. Toxicity analysis with A. cepa signifies that dye Red HE3B exerts oxidative stress and subsequently toxic effect on the root cells where as biodegradation metabolites of the dye are relatively less toxic in nature. Phytotoxicity studies also indicated that microbial treatment favors detoxification of Red HE3B.     

Nitritation and denitritation of ammonium-rich wastewater using fluidized-bed biofilm reactors

Fluidized-bed biofilm nitritation and denitritation reactors (FBBNR and FBBDR) were operated to eliminate the high concentrations of nitrogen by nitritation and denitritation process. The dissolved oxygen (DO) concentration was varied from 1.5 to 2.5 g/m(3) at the top of the reactor throughout the experiment. NH(4)-N conversion and NO(2)-N accumulation in the nitritation reactor effluent was over 90 and 65%, respectively. The average NH(4)-N removal efficiency was 99.2 and 90.1% at the NLR of 0.9 and 1.2 kg NH(4)-N/m(3)day, respectively. Increasing the NLR from 1.1 to 1.2 kg NH(4)-N/m(3)day decreased the NH(4)-N elimination approximately two-fold while NH(4)-N conversion to NO(2)-N differences were negligible. The NO(2)-N/NO(x)-N ratios corresponded to 0.74, 0.73, 0.72, and 0.69, respectively, indicating the occurrence of partial nitrification. An average free ammonia concentration in the FBBNR was high enough to inhibit nitrite oxidizers selectively, and it seems to be a determining factor for NO(2)-N accumulation in the process. In the FBBDR, the NO(x)-N (NO(2)-N+NO(3)-N) concentrations supplied were between 227 and 330 mg N/l (NLR was between 0.08 and 0.4 kg/m(3)day) and the influent flow was increased as long as the total nitrogen removal was close to 90%. The NO(2)-N and NO(3)-N concentrations in the effluent were 3.0 and 0.9 mg/l at 0.08 kg/m(3)day loading rate. About 98% removal of NO(x)-N was achieved at the lowest NLR in the FBBDR. The FBBDR exhibited high nitrogen removal up to the NLR of 0.25 kg/m(3)day. The NO(x)-N effluent concentration never exceeded 15 mg/l. The total nitrogen removal efficiency in the FBBRs was higher than 93% at 21+/-1 degrees C.     

膜生物反应器脱氮除磷工艺处理城市污水的工程应用

为强化城市污水脱氮除磷,研发了厌氧/缺氧/好氧/缺氧-膜生物反应器(A2/O/A-MBR)新工艺,并建设了设计处理规模为2万m3/d实际工程.对该工程的长期监测表明,出水C()D、TN、NH4+-N和TP的平均浓度分别为20.6、6.67、1.05、0.19 mg/L,优于《城镇污水处理厂污染物排放标准》中的一级A标准...     

Ratcheting assessment of materials based on the modified Armstrong–Frederick hardening rule at various uniaxial stress levels

The present study evaluates ratcheting response of materials by means of the Armstrong–Frederick (A–F) hardening rule, the modified A–F rule (Bower's model), and further modifications of the hardening rule based on new introduced coefficients. The implemented modifications on the A–F‐based hardening rule aims to address stages of ratcheting over stress cycles. The modified hardening rule predicts the ratcheting strain rate decay over stage I and the constant rate of strain accumulation during stage II. The modified hardening rule consisted of the coefficients of the hardening rule controlling stress–strain hysteresis loops generated over stress cycles during ratcheting process (Bower's modification on A–F rule) plus the coefficients controlling rates over stages of materials ratcheting deformation. Stress–strain‐dependent coefficients in the modified rule are responsible to compromise overprediction of ratcheting of A–F during stage I and the premature plastic shakedown beyond stage I induced by Bower's model. Ratcheting strain rate coefficients improved the hardening rule capability to calibrate and control the rate of ratcheting in stages I and II and enabled the modified hardening rule to predict ratcheting strain over a prolonged domain of stress cycles. The modified hardening rule was employed to assess ratcheting response of 304, 42CrMo, 316L steel and copper samples under uniaxial loading conditions. The predicted ratcheting values based on the modified hardening rule and the experimental ratcheting strains were found in good agreements.     

Stress-strain diagrams of metals under large uniform compressive strains

A new method was worked out for obtaining compressive stress-strain diagrams for large uniform plastic strains. For the first time correct compressive stress-strain diagrams of copper M2, steels St. 3, 45, 12KhN3A, U8A, and of the high-strength cast iron VCh-50 were obtained. In correct experiments at uniform state of stress the previously suggested postulate of perfect plasticity was confirmed for all the materials without exception. It was established that the compressive stress-strain diagram is described by a parabola fully determined by three fundamental constants, viz., yield point, maximal stress, and the strain at which this is attained. It is shown that for plastic metals the stress-strain diagram for large strains can be predicted from two initial points of the curve, and also from the results of standard tensile tests.Translated from Problemy Prochnosti, No. 9, pp. 53–57, September, 1994.