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1.
摘要:将鸡(Gallus gallus)的γ-干扰素(interferon-γ, IFN-γ)基因亚克隆到杆状病毒转移载体pMelBacB的BamHⅠ位点上,构建真核转移载体pMelBacB-ChIFN-γ,经限制性内切酶消化、ChIFN-γ特异引物PCR鉴定和序列测定,证明目的基因正确克隆到载体的预期位点。将纯化的pMelBacB-ChIFN-γ质粒与杆状病毒DNA(Bac-N-BlueTM DNA)共转染sf9昆虫细胞,经3轮蓝斑筛选纯化,获得了重组杆状病毒rBaculovirus-ChIFN-γ。提取病毒DNA经ChIFN-γ特异引物和重组杆状病毒特异引物PCR鉴定,证明获得了纯化的重组杆状病毒,命名为rBaculovirus-ChIFN-γ。用该重组病毒接种sf9昆虫细胞,收获接种后不同时间的细胞进行SDS-PAGE,结果表明ChIFN-γ基因在昆虫细胞中获得了表达,表达的重组蛋白分子量约为19 kD。应用在大肠杆菌(Escherichia coli )原核表达系统中表达的重组蛋白制备的兔抗ChIFN-γ多克隆抗体进行Western blot分析,表明表达的重组蛋白具有抗原性。  相似文献   

2.
以马立克氏病病毒(MDV)CVI988株基因组为模板,利用PCR技术扩增出约2.7和3.0 kb的基因片段,将上述片段同时插入pUC19中,获得约5.5 kb MDV同源重组臂;以该基因片段的US2区的BglⅡ为插入位点,分别插入基因表达盒CMV-gpt-ployA和CMV-gfp-polyA,构建转移载体pUS-gpt-GFP,将该载体瞬时转染CHO细胞,在荧光显微镜下,可以观察到绿色荧光蛋白的表达;将该转移载体转染已用MDV CVI998株感染的次代CEF细胞,利用MX-HAT培养基筛选重组病毒,并用荧光显微镜挑选表达绿色荧光蛋白的蚀斑,结果获得重组病毒rMDVgptGFP。通过PCR检测和病毒生长测定,证明重组病毒获得纯化。  相似文献   

3.
将人内皮抑素(Endostatin)基因重组于家蚕杆状病毒转移载体pBacPAK8中,获得重组转移载体pBacPAK-Endo,并与修饰性线性化病毒Bm-BacPAK6DNA共转染家蚕培养细胞,二者在细胞内发生内源重组,经蓝白斑筛选及DNA点杂交,鉴定出含有Endostatin基因的重组病毒BmBac-Endo。将重组病毒感染家蚕培养细胞和五龄幼虫,经SDS-PAGE电泳、Western blotting分析及对人血管内皮细胞株ECV304的抑制活性测定,证明Endostatin在家蚕细胞和幼虫中得到高效表达,且表达产物具有良好的生物学活性。  相似文献   

4.
摘要:本实验从质粒pGEM-HA中扩增H5N1亚型禽流感病毒HA基因,构建转移载体pFastBacHT-HA并与E.coliDH10Bac中的Bacmid质粒重组,构建重组转座质粒rBacmid-HA。在脂质体介导下将rBacmid-HA转染sf9昆虫细胞获得重组杆状病毒。在sf9昆虫细胞中表达HA蛋白,通过SDS-PAGE、Western blot和血细胞吸附实验鉴定重组蛋白。结果显示重组HA蛋白分子量约为66kDa,该蛋白与AIV H5亚型鸡血清发生特异性反应,与H7、H9亚型鸡血清不反应,转染重组杆状病毒后的细胞能吸附鸡的红血球,证明HA基因在sf9昆虫细胞中获得了正确表达,不仅具有型特异性,而且有良好的生物反应性。  相似文献   

5.
本研究将PRRSV B13株ORF5基因克隆到杆状病毒转移载体质粒pVL1393中,构建了重组转移载体质粒pVL1393-ORF5.用该重组转移载体质粒与线性化苜蓿丫纹夜蛾核型多角体病毒(AcMNPV-SVI-G)基因组DNA(BaculoGold Linearized Baculovirus DNA)共转染草地夜蛾(Spodoptera frugiperda,sf9)细胞,得到重组病毒AcMNPV-OCC-- ORF5 .在感染了重组病毒的sf9细胞中成功地检测到分子量为25kD的ORF5基因表达产物,其能被猪抗PRRSV B13株多克隆血清特异识别.表明sf9细胞表达了所期望的产物,进而也说明基因片段的正确性.猪抗PRRSV VR-2332株多克隆血清对ORF5基因的表达产物无反应,而猪抗LV株多克隆血清则能特异识别,说明PRRSV中国分离株B13株在抗原性上更接近欧洲亚群分离株LV株的抗原性,进一步证实了PRRSV中国分离株B13株属于欧洲亚群.ORF5基因表达产物与天然蛋白分子量十分接近,说明杆状病毒表达系统能较适当的进行合成蛋白的加工和修饰.本研究的结果为PRRS基因工程疫苗的研究奠定了基础.  相似文献   

6.
恒定链(invariant chain,Ii)是脊椎动物重要的免疫分子,在主要组织相容性复合体(major histocompatibility complex,MHC)Ⅱ类分子递呈抗原中起重要作用。为研究鸡(Gus gallus)Ii与MHCⅡ类分子的关系,本研究构建了4个沉默Ii基因的慢病毒表达载体,并比较了其干扰效果。首先,用自行设计合成的4条鸡Ii基因干扰序列,分别经一步退火法获得双链短发夹RNA(short hairpin RNA,shRNA),并将其用双酶切法插入慢病毒载体pLL3.7。4个构建的慢病毒重组载体经酶切和测序鉴定,分别命名为pLLIi-shRNA236、pLL-Ii-shRNA376、pLL-Ii-shRNA527和pLL-Ii-shRNA539。然后把这些重组载体分别转染293T细胞(转染腺病毒E1A基因(lethal infection gene)的人肾上皮细胞系),进行病毒包装,再用包装的慢病毒感染鸡源巨噬细胞HD-11。结果表明,重组载体感染的293T细胞表达绿色荧光蛋白,用包装的慢病毒接种293T细胞,其滴度达2.2×107~5.1×107TU/mL;接种HD-11细胞的感染率均达到95%以上。最后用荧光定量PCR方法检测其沉默鸡源巨噬细胞HD-11的鸡Ii mRNA的效率,与对照组相比,4个重组慢病毒干扰效率分别为37%、52%、88%和78%;其中,pLL-Ii-shRNA527的干扰效率最高。综上所述,本研究从4个构建的重组载体中筛选出1个高效沉默鸡巨噬细胞Ii基因重组载体,并提示shRNA序列是决定沉默特定基因的关键。实验结果为研究鸡Ii在递呈抗原中与其他免疫分子的关系提供了基础资料。  相似文献   

7.
摘要:利用PCR技术,扩增去除部分序列、长度为1320 bp、编码440个氨基酸的禽流感病毒(AIV)NP基因片段,将其克隆至pR质粒的T4噬菌体SOC基因C末端获得重组质粒pR-NP,以此重组载体转化大肠杆菌E2,用溶菌酶缺陷噬菌体T4-Z1感染重组E2菌,重组载体与缺陷噬菌体T4-Z1基因发生同源重组,将NP基因整合到噬菌体基因组中,用PCR方法筛选重组噬菌体并命名为T4-Z1-NP。经Western blot检测证实,T4-Z1-NP表达的NP融合蛋白具有免疫学活性。成功构建了表达禽流感病毒核蛋白的重组噬菌体。  相似文献   

8.
Dvl1及其结构缺失突变体ΔDIX(dsh and axin)和ΔDEP(dsh,Egl-10 and pleckstrin)腺病毒载体的构建并验证其在MSCs(mesenchymal stem cells)中的表达情况。将目的基因定向克隆至pAdTrack-CMV穿梭载体上,并经PmeⅠ线性化后在BJ5183细菌中与pAdEasy-1骨架质粒同源重组,获得重组腺病毒载体,在QBI-293A细胞中包装及扩增,实时荧光定量PCR和Western bolt验证Dvl1在MSCs中的表达情况。经PCR、PacⅠ单酶切鉴定及测序分析,成功构建Ad-Dvl1、Ad-ΔDIX和Ad-ΔDEP腺病毒载体,目的基因序列与GenBank报道一致,并选出150 MOI为最适感染MSCs的感染复数,成功构建了Ad-Dvl1、Ad-ΔDIX和Ad-ΔDEP腺病毒载体,并获得高滴度的病毒子,qPCR和Western blot证实Dvl1在MSCs中高表达并增加了β-catenin在细胞中的累积,为进一步研究Dvl1在MSCs迁移过程的作用奠定了基础。  相似文献   

9.
奶牛γ-干扰素基因的克隆及其在COS-1细胞中的表达   总被引:3,自引:0,他引:3  
应用RT—PCR技术从经体外植物血凝素(PHA)刺激诱导的奶牛外周血淋巴细胞的总RNA中扩增出牛γ-干扰素(bovine interferon-γ,BovIFN-γ)基因。将扩增出的BovIFN-γ克隆到PMD18-T载体中,经过限制性酶切分析筛选正向插入的克隆。测序结果表明,所克隆到的基因编码区序列与已报道基因存在3个核苷酸的差异。将该基因片段从PMD18-T载体中用Hind Ⅲ和BamH Ⅰ双酶切后克隆到真核表达载体pcDNA3.1/zeo ( )中,构建真核表达载体pcDNA—BovIFN-γ,通过脂质体转染COS-1细胞,用鼠抗BovIFN-γ高免血清和FITC标记的羊抗鼠IgG进行间接免疫荧光实验(IFA)检测,结果表明,在COS—1细胞的细胞膜和胞浆内均有rBovIFN-γ表达,细胞病变抑制实验证实,转染后细胞培养上清液具有一定的干扰素活性,转染后72h细胞培养上清液在MDBK细胞上抑制VSV病毒的效价可达到128IU/mL。研究结果为进一步筛选稳定表达BovIFN-γ基因细胞系、开发奶牛疾病预防控制的新产品以及开展奶牛疾病的基因治疗奠定了基础。  相似文献   

10.
猪圆环病毒2型BF株ORF2基因在大肠杆菌中的表达   总被引:6,自引:0,他引:6  
采用PCR从构建的猪圆环病毒2型/(PCV2)ORF2重组质粒(pGEM-T-ORF2)中扩增出大小为593bp的ORb2基因,克隆到表达载体pET-32a,经异丙基硫代半乳糖苷诱导,成功表达了ORF2基因编码的结构蛋白。表达的重组蛋白为融合蛋白,分子量40kD,表达量20%左右。经Western blot检测,重组蛋白可被PCV2阳性血清识别。  相似文献   

11.
【目的】以富士(Fuji)、 秦冠(Qinguan)嫁接在平邑甜茶(Malus hupehensis Rehd.)上的当年生盆栽苗为试验材料,采用砂培方法,研究了缺氮胁迫和干旱对富士和秦冠生长情况、 光合参数、 植株各部位氮磷钾含量及氮素利用效率的影响,分析比较了低氮干旱条件下富士和秦冠生长及氮素利用的差异,以期为果树生产高效肥水利用提供理论指导。【方法】试验共设四个处理: 正常氮正常水(ZZ)、 低氮正常水(DZ)、 正常氮干旱(ZG)、 低氮干旱(DG)。氮素和水分均设置两个水平,分别为正常氮(6 mmol/L NO-3-N)、 低氮(0.3 mmol/LNO-3-N)、 正常供水(保持盆中砂子相对含水量为饱和含水量的80%~85%)、 干旱处理(保持盆中砂子相对含水量为饱和含水量的60%~65%)。【结果】富士和秦冠的生物量(茎和叶)、 株高茎粗等生长指标以及光合速率、 气孔导度、 蒸腾速率均为正常氮正常水(ZZ)>低氮正常水(DZ)>正常氮干旱(ZG)>低氮干旱(DG),并且相对应处理下秦冠的以上指标均高于富士;正常供水下,缺氮处理使富士、 秦冠的根冠比比正常氮处理均有所增加,富士提高了2.05%,秦冠提高了22.40%。富士和秦冠的氮、 磷、 钾含量均表现出正常氮正常水(ZZ)>低氮正常水(DZ)>正常氮干旱(ZG)>低氮干旱(DG); 氮、 钾元素含量在植株各部位的分布顺序依次是叶>根>茎,磷元素则是根>叶>茎;光合氮素利用效率(PNUE)和氮素利用效率表现为秦冠处理之间差异极显著,富士处理之间差异不显著;秦冠的PNUE和NUE明显高于富士,在低氮正常水(DZ)处理下,秦冠氮肥利用率比富士高42.07%,在低氮干旱(DG)处理下高64.14%;低氮胁迫下富士和秦冠的NUE显著提高,并且秦冠提高的幅度高于富士。【结论】施用氮肥能够显著提高富士与秦冠的干物质量,同等水肥条件下,秦冠生长优于富士;水分亏缺会减少叶片对氮的吸收,干旱条件下适度增施氮肥,可提高果树的抗旱能力;低氮干旱胁迫下秦冠的生长指标、 光合指标及氮素利用效率指标均优于富士,表现出较强的抗低氮干旱胁迫的能力。  相似文献   

12.
Laser-induced breakdown spectroscopy (LIBS) is a new technique for the analysis of plant material. This study investigates the application of LIBS to pasture-based plant samples. The LIBS measurements were obtained from pelletized pasture samples (100 samples) that had been also analyzed by inductively coupled plasma–optical emission spectroscopy (ICP-OES) following microwave digestion for calibration and comparison purposes. Comparisons for elements sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), manganese (Mn), iron (Fe), copper (Cu), zinc (Zn), boron (B), phosphorus (P), and sulfur (S) showed that LIBS could be used for almost all the standard profile total elements with concentrations down to low mg/kg levels (observed error of Na: 0.024 percent, K: 0.18 percent, Mg: 0.016 percent, Ca: 0.073 percent, P: 0.017 percent, Mn: 31 mg/kg, Fe: 150 mg/kg, Zn: 6.6 mg/kg, and B: 1.1 mg/kg). Elemental analysis at less than mg/kg levels was not possible using LIBS. The elements S and Cu were particularly difficult to analyze with reliability using LIBS at the concentration levels found in the plant samples. Replacing microwave digestion and subsequent ICP analysis with a direct analysis of dried plant samples using LIBS has the potential to improve the productivity and reduce the cost of testing.  相似文献   

13.
正The Center for Agricultural Resources Research(CARR),the Institute of Genetics and Developmental Biology(IGDB),Chinese Academy of Sciences,invites applicants for several research group leader positions.CARR is one of the research organizations in Chinese Academy of Sciences(CAS).We seek nominations and applications from individuals who have expertise and a record of accomplishment in research areas related to ecology,agro-hydrology,  相似文献   

14.
The responses of tallgrass prairie plant communities and ecosystem processes to fire and grazing are well characterized. However, responses of invertebrate consumer groups, and particularly soil-dwelling organisms, to these disturbances are not well known. At Konza Prairie Biological Station, we sampled soil macroinvertebrates in 1994 and 1999 as part of a long-term experiment designed to examine the effects and interactions of annual fire, mowing, and fertilization (N and P) on prairie soil communities and processes. For nearly all taxa, in both years, responses were characterized by significant treatment interactions, but some general patterns were evident. Introduced European earthworms (Aporrectodea spp. and Octolasion spp.) were most abundant in plots where fire was excluded, and the proportion of the total earthworm community consisting of introduced earthworms was greater in unburned, unmowed, and fertilized plots. Nymphs of two Cicada genera were collected (Cicadetta spp. and Tibicen spp.). Cicadetta nymphs were more abundant in burned plots, but mowing reduced their abundance. Tibicen nymphs were collected almost exclusively from unburned plots. Treatment effects on herbivorous beetle larvae (Scarabaeidae, Elateridae, and Curculionidae) were variable, but nutrient additions (N or P) usually resulted in greater densities, whereas mowing usually resulted in lower densities. Our results suggest that departures from historical disturbance regimes (i.e. frequent fire and grazing) may render soils more susceptible to increased numbers of European earthworms, and that interactions between fire, aboveground biomass removal, and vegetation responses affect the structure and composition of invertebrate communities in tallgrass prairie soils.  相似文献   

15.
Abstract

A 3-year study was carried out to investigate quality parameters in 14 tree fruit and berry species grown in southern Norway. The species were blueberry, apple, aronia, sour cherry, sweet cherry, red raspberry, strawberry, blackcurrant, gooseberry, red currant and elderberry, harvested along with wild bilberry, cloudberry and lingonberry. Significant differences between species were identified for all quality parameters. The coefficient of variation between species was lowest for pH (12.5%), dry matter (18.9%) and soluble solids (25.3%), followed by titratable acids (59.3%), total phenolics (83.8%), antioxidant capacity FRAP (85.7%) and antiradical power by the DPPH-assay (97.8%), total monomeric anthocyanins (132%) and ascorbic acid (137%). Average coefficient of variation within species were lower and ranged from 4 (pH) to 62% (ascorbic acid). Only the FRAP values were significantly affected by harvesting year with lower levels in 2004 than in 2005 and 2006. There were significant interactions between species and harvesting year for dry matter, soluble solids, pH, ascorbic acid and FRAP. The results indicate generic ranges in composition within species independent upon growing location and climate, and the composition of the tree fruits and berries is not likely to deviate from these ranges. It is concluded that desirable composition of tree fruits and berries and their products should primarily be achieved by selection among species rather than searching fors broadened variation within individual species.  相似文献   

16.
Potassium (K) fixation and release in soil are important factors in the long-term sustainability of a cropping system. Changes in K concentration and characteristics of K fixation and release in rhizosphere and nonrhizosphere soils in the rapeseed (Brassica napus L.)–rice (Oryza sativa L.) rotation were investigated using a rhizobox system. The concentrations of different forms of K in both rhizosphere and nonrhizosphere soils decreased with plants compared to without plants, regardless of K fertilizer application. Potassium uptake by crops mainly came from the rhizosphere soil. In the treatment without K fertilizer (–K), the main form of K supplied by the soil to the crops was 1.0 mol L?1 nitric acid (HNO3) nonextractable K, followed by nonexchangeable K, and then exchangeable K. In the treatment with K fertilizer (+K), the main K forms supplied by the soil to the crops were exchangeable K and nonexchangeable K. The amount and rate of K fixation after one cycle of the rapeseed–rice rotation was greater in rhizosphere soil than in nonrhizosphere soil. The amount and rate of K fixation of soil in the +K treatment were significantly less than in the –K treatment. The cumulative amounts of K released with 1.0 mol L?1 ammonium acetate (NH4OAc) and 1.0 mol L?1 HNO3 extraction increased with the increasing numbers of extractions, but the K-releasing power of soil by successive extraction decreased gradually and finally became almost constant. The release of K was less in rhizosphere soil than in nonrhizosphere soil. The release of K in the +K treatment was similar to that in the –K treatment in rhizosphere soil, but the K release in nonrhizosphere soil was greater with the +K than the –K treatment. Overall, the information obtained in this study will be helpful in formulating more precise K fertilizer recommendations for certain soils.  相似文献   

17.
Inoculation of wheat seedlings with the plant growth-promoting bacterium Azospirillum brasilense Cd was immobilized in alginate microbeads and, without applying any stress, significantly increased the quantity of several photosynthetic pigments, such as chlorophyll a, chlorophyll b, and the auxiliary photoprotective pigments violaxanthin, zeaxanthin, antheroxanthin, lutein, neoxanthin, and β-carotene. This resulted in greener plants with no apparent visible stress. After monitoring the quantity of photosynthetic pigments for 4 weeks, we observed that inoculated plants had higher quantities of pigments in shoot and stem. The greatest difference in the quantity of all pigments between inoculated and noninoculated plants occurred in the first week of growth. Regardless of treatment, the quantity of pigments in stems was three to four times less than the quantity of these pigments in shoots. Application of Azospirillum, either as liquid inoculant or as alginate microbeads, did not alter the positive effect of the bacteria on pigment production or the positive response of the plants towards A. brasilense Cd inoculation.  相似文献   

18.
We examined the community composition of microbes that colonized atrazine-containing beads buried in agricultural soils that differed in atrazine treatment history. Bacterial abundance was 5-40-fold greater in atrazine-fortified beads. In beads containing 20 mg atrazine kg−1 buried in soil with a history of atrazine application (conditioned soil), the abundance of Actinobacteria increased approximately 80-fold whereas in control soil, Actinobacteria were enriched only 10-fold and the gamma-Proteobacteria and Planctomycetes increased by 60- and 25-fold, respectively. The gamma-Proteobacteria were enriched by 120- and 230-fold in beads containing 200 mg atrazine kg−1 in conditioned and control soil, respectively. The results demonstrate that BioSep® beads are a suitable matrix for recruiting a diverse subset of the bacterial community involved in atrazine degradation.  相似文献   

19.
Arbitrary oligonucleotides were used as primers to amplifygenomic DNA of 48 wild Spanish populations of Agropyroncristatum, Elymus hispanicus,E. caninus,E. repens,Thinopyrum curvifolium, Th.junceum and Th.intermedium. Genetic diversity was analysedusing nineteen primers. The number of amplified products ranged from8 to 18 per primer and a total of 240 markers were scored. Differentlevels of intraspecific genetic diversity were found, the allogamousspecies E. repens andTh. intermedium being themost variable. Jaccard's similarity coefficients for internalmeasure within and between populations were used to produce a clusterdiagram. The results demonstrate differences in the degree ofsimilarity between taxonomic units. Interpopulational variability andinterspecific genomic relationships of these species arediscussed.  相似文献   

20.
The effects of three commonly used fungicides on the colonization and sporulation by a mixture of three arbuscular mycorrhizal (AM) fungi consisting of Glomus etunicatum (Becker & Gerd.), Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe, and Gigaspora rosea (Nicol. & Schenck) in symbiosis with pea plants and the resulting response of the host-plant were examined. Benomyl, PCNB, and captan were applied as soil drenches at a rate of 20 mg active ingredient kg-1 soil 2 weeks after transplanting pea seedlings in a silty clay-loam soil containing the mixed inocula of AM fungi (AM plants). Effects of fungicides were compared to untreated plants that were inoculated with fungi (AM control). The effect of mycorrhizal inoculation on plant growth was also examined by including nonmycorrhizal, non-fungicide-treated plants (non-AM control). Fungicides or inoculation with AM fungi had only a small effect on the final shoot weights of pea plants, but had greater effects on root length and seed yield. AM control plants had higher seed yields and lower root lengths than the corresponding non-AM plants, and the fungicide-treated AM plants had intermediate yields and root lengths. Seed N and P contents were likewise highest in AM control plants, lowest in non-AM plants, and intermediate in fungicide-treated AM plants. All three fungicides depressed the proportion (%) of root length colonized by AM fungi, but these differences did not translate to reductions in the total root length that was colonized, since roots were longer in the fungicide-treated AM plants. Pea plants apparently compensated for the reduction in AM-fungal metabolism due to fungicides by increasing root growth. Fungicides affected the population of the three fungi as determined by sporulation at the final harvest. Captan significantly reduced the number, relative abundance, and relative volume of G. rosea spores in the final population relative to the controls. The relative volume of G. etunicatum spores was greater in all the fungicide-treated soils, while G. mosseae relative volumes were only greater in the captan-treated soil. These findings show that fungicides can alter the species composition of an AM-fungal community. The results also show that AM fungi can increase seed yield without enhancing the vegetative shoot growth of host plants.  相似文献   

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