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1.
基因芯片(gene chip)是依据核酸杂交原理发展的一种生物新技术,在生命科学研究领域具有重要的应用价值.本研究将不对称PCR和基因芯片两种技术相结合,构建了同步检测鸡(Gallus gallus)传染性喉气管炎病毒(Infectious laryngotracheitis virus,ILTV)、新城疫病毒(Newcastle disease virus,NDV)、传染性支气管炎病毒(Infectious bronchitis virus,IBV)的共检基因芯片.分别选取ILTV的胸苷激酶(thymidine kinase,TK)和糖蛋白B(glycoproteins B,gB)基因、NDV的融合蛋白(fusion,F)和血凝素-神经氨酸酶蛋白(haemagglutinin-neuraminidase,HN)基因以及IBV的膜蛋白(membrane,M)和核衣壳(nucleocapsid,N)基因设计引物,从重组质粒菌中扩增制备探针基因,用乙醇沉淀法纯化后点制于氨基修饰的载玻片上,制备基因芯片;靶基因用cy3标记引物,进行不对称PCR扩增,扩增的荧光标记单链产物与芯片杂交.不对称PCR结果显示,当限制性引物与非限制性引物浓度比例在1:10时ILTV-TK、NDV-HN和IBV-N的单链产物增加最多,当浓度比在1:20时,ILTV-gB、NDV-F和IBV-M的单链产物增加最多;相应的标记样品与3种病毒检测芯片杂交后,均出现较强的杂交信号,而阴性对照检测不到荧光信号,灵敏性实验表明,当DNA浓度为1.8x 104拷贝时杂交仍为阳性.本研究构建的诊断基因芯片对12份临床样品进行初步应用检测,与PCR检测技术检出率基本一致.本实验所建立的联合检测基因芯片能够快速、准确、高通量的诊断NDV-IBV-ILTV,可以应用于集约化养殖业中对多种鸡疫病病毒的检测. 相似文献
2.
猪流行性腹泻病毒纤突蛋白基因克隆与序列分析 总被引：2，自引：0，他引：2
对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)DX株纤突蛋白基因进行了克隆与测序。结果表明,该基因核苷酸序列长为4152bp,推导氨基酸序列为1383aa,与比较毒株氨基酸同源性都在90%以上。系统发生树分析结果表明,比较毒株可分为3个基因群,而且每个群的毒株均来自同一个国家。利用生物软件分析结果表明PEDV发生微小的变异,尤其在韩国变异明显,而DX株与中国JS-2004-2株亲缘关系很近。 相似文献
3.
利用基因重组技术将鸡传染性支气管炎病毒（IBV）S1基因分段（A和B段）克隆到pGEX—6p-1载体中，用IPTG诱导表达。SDS-PAGE电泳结果显示有明显的融合蛋白表达条带。通过IBV特异性抗体的Western blot检测，两段表达产物均能与抗体发生特异性反应，证明表达产物具有部分抗原性。用Glutathione Sepharose 4B纯化出A段和B段的GST融合蛋白。将纯化得到的融合蛋白免疫家兔。连续免疫3次，将得到的抗体与200 TOCID50（气管环半数感染）/0．1mL病毒工作液在37℃温育1h进行TOC试验。经计算A段抗体的50%血清中和终点为1：18，而B段抗体的50％血清中和终点为1：53，表明这两段的表达产物具有较好的免疫原性，B段表达产物更具有良好的抗原性，适合作为免疫抗原和诊断抗原，具有一定的临床应用价值。 相似文献
4.
家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)bro (baculovirus repeated ORF)-d基因普遍存在于多种感染鳞翅目昆虫的多角体病毒基因组中,是杆状病毒中一类重复开放阅读框序列.为了解bro-d基因在病毒感染过程中的作用及其与宿主细胞凋亡之间的关系,本研究利用Red重组技术敲除BmNPV基因组中的bro-d基因,构建基因缺失型病毒bro-d-ko-Bacmid,并将该缺失型病毒转染家蚕BmN细胞,利用qRT-PCR技术检测bro-d基因的缺失对病毒基因组复制、转录水平以及抑制凋亡蛋白2基因(inhibitor of apoptosis protein 2,iap2)的影响.结果显示,bro-d基因缺失后会导致病毒基因组的复制水平显著下降(P＜0.05),同时病毒的早期基因lef-3、晚期基因vp39和极晚期基因p1O的转录水平也都显著下降(P＜0.05);iap2的转录水平显著下调(P＜0.05).在bro-d基因缺失型病毒的复制水平明显低于野生型病毒的情况下,仍会导致细胞活力显著下降(P＜0.05);bro-d基因缺失引起细胞B淋巴细胞瘤-2(B-celllymphoma-2,Bcl-2)蛋白含量明显低于野生型病毒,而Bcl-2相关X蛋白(Bcl-2 assaciated X protein,Bax)含量显著高于野生型病毒(P＜0.05),进而导致细胞凋亡水平上升.结果表明,bro-d基因不仅对病毒各时期基因表达水平具有调控作用,也可通过调控iap2的表达进而调控宿主细胞的凋亡水平.研究成果为深入了解bro-d基因在病毒感染过程中的功能提供了基础资料,也为通过延长宿主细胞寿命、提高目的蛋白表达产量来进行杆状病毒表达载体的改造提供了新思路. 相似文献
5.
本研究应用鸡胚尿囊腔接种的方法,从广西患传染性支气管炎的免疫失败的病鸡中分离到一株传染性支气管炎病毒（IBV）（命名GX-YL5）,通过间接血凝试验、动物回归试验和气管环交叉中和试验对该毒株进行鉴定和主要生物学特性的研究,同时利用RT-PCR技术扩增克隆该病毒的S1基因和N基因并测定其核苷酸序列。结果表明：该病毒株有间接血凝性;致病性较强;血清型不同于常用疫苗株H120、Ma5、4／91。同源性分析表明,S1基因核苷酸和氨基酸序列与参考株的同源性分别为63.5％-81.2％和50.7％-78.8％;N基因核苷酸和氨基酸序列与参考株的同源性分别为85.7％-87.2％和89.5％-91.7％;S1基因和N基因系统进化分析表明分离株与其它参考毒株的亲缘关系较远。S1基因和N基因分型与血清学试验结果相吻合。本研究结果提示了广西分离株GX-YL5可能是一个新的变异株,这可能是目前免疫失败的重要因素,同时也为研制适合本地使用的IBV疫苗提供了科学依据和物质基础。 相似文献
6.
传染性支气管炎病毒S1纤突糖蛋白基因的克隆及部分序列分析 总被引：4，自引：1，他引：3
大纤突Ｓ糖蛋白是传染性支气管炎病毒重要的免疫相关性蛋白之一，参考已发表的ＩＢＶ－Ｂｅａｕｄｅｔｔｅ株Ｓ１基因ＤＮＡ序列，合成一对特异性引物，以ＩＢＶ－ＲＮＡ为模板，应用ＲＴ－ＰＣＲ方法，获得传染性支气管炎病毒上海分离株Ｓ２Ｔ２－Ｓ１纤突糖蛋白基因，长度１．６５ｋｂ，利用引物设计中的ＢａｍＨ和ＨｉｎｄⅢ位点将其克隆到载体ｐＳＩ（＋）中。对该基因进行限制性酶发分析，结果与已报道的相一致。 相似文献
7.
鸡传染性支气管炎病毒嗜腺胃毒株（ZJ971）核衣壳蛋白基因的克隆 总被引：2，自引：0，他引：2
8.
李建强 《农业生物技术学报》2007,15(4)
摘要:对猪源冠状病毒PEDV DX株纤突蛋白基因进行了克隆与测序。结果表明该基因核苷酸序列长为4152 nt, 推导氨基酸序列为1383aa，与比较毒株氨基酸同源性都在90%以上。系统发生树分析结果表明，比较毒株可分为3个基因群，而且每个群的毒株均来自同一个国家。利用生物软件分析结果表明PEDV发生着微小的变异，尤其在韩国变异明显，而DX株与中国JS-2004-2株亲缘关系很近。 相似文献
9.
检测四种鸡呼吸道疫病病毒可视化芯片技术的优化及初步应用 总被引：1，自引：0，他引：1
可视化芯片技术是在传统芯片技术的基础上发展起来的一项新的疾病诊断和基因分析技术,对于临床疾病检测和诊断具有重要意义。本研究针对禽流感病毒(Avian influenza virus,AIV)的核蛋白(nucleprotein,NP)基因设计一对特异性引物,以H9亚型禽流感病毒分离株c DNA为模板,经PCR扩增、连接、转化和核酸序列鉴定后,得到含有AIV-NP基因的重组质粒。同时复苏本实验室保存的3株分别含有新城疫病毒(Newcastle disease virus,NDV)的融合(fusion,F)蛋白基因、鸡传染性喉气管炎病毒(Infectious laryngotracheitis virus,ILTV)胸苷激酶(haemaggluttinin-neuraminidase,TK)基因和鸡传染性支气管炎病毒(Infectious bronchitis virus,IBV)核衣壳(nucleocapsid,N)蛋白基因的重组菌。以上述4种病原的靶基因核酸序列片段的正义链为模板,设计寡核苷酸探针,喷样到尼龙膜上制备成芯片,利用不对称PCR技术扩增生物素标记的靶基因,与芯片进行杂交后,检测结果直接用肉眼就可以判定。本研究对芯片制备流程和检测过程中主要条件进行优化。结果表明当寡核苷酸探针喷样浓度为25μmol/L、芯片杂交反应的时间为1 h、杂交温度为50℃、Streptavidin HRP Conjugate(1.0 mg/m L)稀释2 000倍、二氨基联苯胺(diaminobenzidine,DAB)显色时间为5 min时,芯片检测技术的结果最佳。用该方法与PCR/RT-PCR技术同时对临床采集的96份疑似病料进行检测,两种方法的检测结果一致。本研究构建的检测4种禽呼吸道疾病病毒可视化基因芯片技术具有高通量、快速、准确等优点,为鸡病的临床诊断提供了新的技术。 相似文献
10.
从2005~2006年中国部分地区鸡场疑似鸡传染性支气管炎病毒(Avian infectious bronchitis virus)感染鸡分离到12株鸡传染性支气管炎病毒,并对其部分生物学特性及基因型进行了研究。发现其中11株病毒分属于LX4和CK/CH/LSC/99Ⅰ型。通过S1基因比较、进化树分析、BLAST搜寻和动物试验研究发现,CK/CH/LSD/05Ⅰ是一株新型变异毒株。动物感染试验表明,CK/CH/LSD/05Ⅰ感染鸡的肾脏病变不明显,仅有20%的感染鸡肾脏病毒分离呈阳性,但100%感染鸡呼吸道病毒分离阳性,表明该病毒不是中国普遍流行的肾型毒株,而其对呼吸道具有较强嗜性。同时,实验应用弱毒疫苗H120和本实验室致弱的3株异种毒株进行了动物保护试验,结果显示,用CK/CH/LSD/05Ⅰ攻毒后,这4株异种传染性支气管炎病毒的免疫对气管的保护率很低。初步揭示该毒株可能属新的血清型病毒。 相似文献
11.
In this study, data on cattle depredation by puma (Puma concolor) and jaguar (Panthera onca) were recorded for six years (1998-2003) in a cattle ranch in central-western Brazil. Depredation represented 18.9% of the overall cattle mortality, being predominant on calves. In biomass, kills represented 0.4% (63.8 kg/km2) of the ranch’s annual stock. In economic loss, kills represented 0.3% of the cattle stock value. Depredation was mainly associated with cattle’s age class and location along with the time of birth of calves. The proportion of pastures next to forest with depredation (n = 33, 48.5%) was not distinguished to the proportion of pastures not bordering forest with depredation (n = 35, 51.5%). However, the proportion of pastures next to forest with depredation represented 54% (n = 33) of the 61 total pastures that were at least partially surrounded by forest patches or riparian forests that comprised eight continuum blocks of forest fragments of different sizes in the ranch and adjacent areas. No kills occurred in the central portion (main house) of the farm, close to the headquarters where the pastures not bordering forest. The distances of the kills in relation to areas of native forest was 1317.48 ± 941.03 m. In order to reduce depredation, calves should be kept as far as possible from forest areas and concentrated cattle breeding and calving seasons should be encouraged. 相似文献
12.
Decomposer animals stimulate plant growth by indirect effects such as increasing nutrient availability or by modifying microbial communities in the rhizosphere. In grasslands, the spatial distribution of organic matter (OM) rich in nutrients depends on agricultural practice and the bioturbation activities of large detritivores, such as earthworms. We hypothesized that plants of different functional groups with contrasting nutrient uptake and resource allocation strategies differentially benefit from sites in soil with OM accumulation and the presence of decomposer animals. In a greenhouse experiment we investigated effects of spatial distribution of 15N-labelled grass litter, earthworms and collembola on a simple grassland community consisting of Lolium perenne (grass) and Trifolium repens (legume). Litter aggregates (compared to homogeneous litter distribution) increased total shoot biomass, root biomass and 15N uptake by the plants. Earthworms and collembola did not affect total N uptake of T. repens; however, the presence of both increased 15N uptake by T. repens and L. perenne. Earthworms increased shoot biomass of T. repens 1.11-fold and that of L. perenne 2.50 fold. Biomass of L. perenne was at a maximum in the presence of earthworms, collembola and with litter concentrated in a single aggregate. Shoot biomass of T. repens increased in the presence of collembola, with L. perenne generally responding opposingly. The results indicate that the composition of the decomposer community and the distribution of OM in soil affect plant competition and therefore plant community composition. 相似文献
13.
Marina Barilani 《Biological conservation》2007,137(1):57-69
The decline of over-hunted red-legged (Alectoris rufa) and rock (A. graeca) partridge populations has been contrasted with massive releases of captive-reared birds, often hybrids with non-indigenous A. chukar. Released interspecific hybrids raise the risks of introgressive hybridisation, and can contribute to further depress the fitness of native populations. Aiming to assess the extent of hybridisation, we genotyped the mtDNA control-region and eight nuclear microsatellites in 671 red-legged, rock and chukar partridges and hybrids, identified by phenotypic traits. Results reveal a diffuse occurrence of hybridisation: (1) 39 samples (6.2%) show mtDNA haplotypes discordant with their phenotypes, indicating red-legged and chukar mtDNA introgression in native rock partridges; (2) admixture analyses of the microsatellite genotypes identified 32 additional rock partridges (5.1%) hybridised mainly with chukars. We analysed also 39 samples collected from a presumed natural red-legged x rock partridge hybrid zone in the French Alps. Surprisingly, 28% birds showed typical chukar mtDNAs, indicating hybridisation with introduced chukars or hybrids. This hybrid zone led to an introgression cline of chukar alleles into neighbouring Alpine rock partridges detectable up to 100 km, which was shorter than expected by neutral genetic theory, and that suggested natural selection against hybrids. These findings indicate that introgressive hybridisation may disrupt local adaptations in natural red-legged partridge and rock partridge populations, and call for strict control of farming and restocking operations. 相似文献
14.
S.P Carter 《Biological conservation》2003,111(3):371-376
The water vole has recently undergone a catastrophic decline in the UK. This has resulted from loss and fragmentation of suitable habitat leaving water vole populations highly vulnerable to the impact of predation by introduced American mink. However, at some reedbed sites water voles and mink have apparently coexisted for many years. To determine if reedbeds offer a refuge from predation, 70 voles were radio-tagged at three sites in England and overwinter mortality monitored. Water vole perception of predation risk was also assessed. Mortality was high (64%), predation by mustelids, including mink, being the chief cause. Experiments suggested that voles failed to perceive areas of highest predation risk. However, predation rate declined strongly with the distance water voles lived from a main water channel. Thus, reedbeds provide a refuge from predation, even by mink, and calculations suggest that they may support source populations enhancing the viability of water vole metapopulations. Consequently reedbeds are now being used as one focus for the conservation of water voles in England and Wales. 相似文献
15.
European honey bees used for commercial honey production represent a potential source of competition for floral resources with native nectar and pollen feeding insects. This study reports the results of an experiment run over two years on the impact of commercial honey bees on the fecundity of a solitary native bee, Hylaeus alcyoneus. Registered apiary sites were used as treatment sites (with honey bees) while control sites (without honey bees) were interspersed between. The fecundity of H. alcyoneus was measured using trap nests. We compared the number of nests produced, number of eggs per nest and emerging progeny mass of H. alcyoneus in sites with and without commercial bee hives. The number of nests produced by H. alcyoneus was 23% less (Wilcoxon’s T) at treatment sites than control sites. Analysis of individual measurement intervals using ANOVA was compromised by a general lack of power. This result highlights that even though honey bees have been present in certain areas for many years, competition with native bees may still be occurring. 相似文献
16.
Graham Robertson Malcolm McNeill Barbara Wienecke Frederique Olivier 《Biological conservation》2006,132(4):458-471
Longline fisheries have been responsible for the deaths of large numbers of seabirds worldwide. Two of the most difficult seabird species to deter from baited hooks are the white-chinned petrel (Procellaria aequinoctialis) and sooty shearwater (Puffinus griseus). Longlines with integrated weight (IW) sink faster than normal, unweighted (UW), longlines and have the potential to reduce the numbers of these species killed. The relative differences in the number of white-chinned petrels and sooty shearwaters killed on UW longlines and IW longlines containing 50 g/m beaded lead core were investigated in 2002 and 2003 in the New Zealand ling (Genypterus blacodes) autoline fishery. Effects on catch rates of target and non-target fish species, and operational aspects of the use of IW gear, were also assessed. A single bird scaring streamer line was deployed on all sets of longlines. In 2002 and 2003, compared to UW lines IW lines reduced mortality of white-chinned petrels by 98.7% and 93.5%, respectively. In 2003 IW lines reduced sooty shearwater mortality by 60.5%. Catch rates of white-chinned petrels (including on sets when no fatalities were recorded) by IW gear were low: 0.005/1000 hooks (UW: 0.4) and 0.01/1000 hooks (UW: 0.17) in 2002 and 2003, respectively. Equivalent rates for sooty shearwaters in 2003 were 0.06/1000 hooks by IW and 0.13/1000 hooks by UW. No albatrosses were caught on either line type in 2002; in 2003 a single Salvin’s albatross (Thalassarche salvini) was caught on UW gear. Both the number of ling and the mean masses of ling caught in 2003 by IW (mass: 189.3; CL95% 162.7-220.3 kg/1000 hooks) and UW (200.6; CL95% 178.2-225.9 kg/1000 hooks) longlines were similar statistically. Use of IW longlines and streamer lines in autoline fisheries should yield major conservation benefits to seabirds interacting with these fisheries worldwide. 相似文献
17.
Two strains of Gluconacetobacter diazotrophicus (Pal 5, UAP5541) and the arbuscular mycorrhizal fungus Glomus intraradices increased both the shoot and root dry weight of sorghum 45 days after inoculation, whereas they had no effect on the shoot and root dry weight of maize. Co-inoculation (Gluconacetobacter diazotrophicus plus Glomus mosseae) did not increase the shoot and root dry weight of either plant. There was a synergistic effect of Gluconacetobacter diazotrophicus on root colonization of maize by Glomus intraradices, whereas an antagonistic interaction was observed in the sorghum root where the number of Gluconacetobacter diazotrophicus and the colonization by Glomus intraradices were reduced. Plant roots inoculated with Gluconacetobacter diazotrophicus and Glomus intraradices, either separately or together, significantly increased root endoglucanase, endopolymethylgalacturonase and endoxyloglucanase activities. The increase varied according to the plant. For example, in comparison with non-inoculated plants, there were higher endoglucanase (+328%), endopolymethylgalacturonase (+180%) and endoxyloglucanase (+125%) activities in 45-day old co-inoculated maize, but not in 45-day old sorghum. The possibility is discussed that hydrolytic enzyme activities were increased as a result of inoculation with Gluconacetobacter diazotrophicus, considering this to be one of the mechanisms by which these bacteria may increase root colonization by AM fungi. 相似文献
18.
Neil Bayfield 《Biological conservation》1979,16(3):219-232
Numbers of Molophilus ater (Meigen) (Diptera, Tipulidae) were lower in trampled peat along a footpath than in adjacent untrampled ground. Peat cores transplanted from the path into undisturbed ground subsequently contained similar numbers of larvae to undisturbed areas, but cores from two larger trampled areas where there was no further disturbance contained fewer larvae; possibly fewer eggs were laid there, or survival was poor. Laboratory observations showed that M. ater imagines tended to spend more time on vegetated ground than bare ground, although egg laying was recorded more often on bare ground; large bare areas might, however, be less attractive. Trampling experiments indicated that physical crushing could kill a high proportion of larvae in peat cores, particularly those near the surface. It was concluded that the low numbers of larvae on the path could be explained by a combination of physical crushing and possibly smaller numbers of eggs or poor survival. 相似文献
19.
Ring-necked parakeets (Psittacula krameri) originate from Africa and Asia, but have been widely introduced and are thought to compete for nest-sites with native hole-nesting birds, raising concerns for the loss of native avifauna. In this study, we adopt a replicated before-after-control experimental design to test for competition between parakeets and native cavity-nesters. In experimental plots in Brussels, Belgium we blocked parakeet nesting cavities and conducted surveys to evaluate availability and use of cavities by parakeets and native hole-nesters. Numbers of breeding pairs, as well as cavity quality index - based on cavity occupancy rates - were evaluated before and after blocking. After blocking, numbers of native nuthatches (Sitta europaea) declined significantly, largely due to nest take-overs by parakeets. Nuthatches and parakeets strongly overlap in preferred nest-sites and this overlap explains the competition that leads to a reduction in nuthatch numbers when suitable cavities become scarce. Average quality of parakeet nests was lower after blocking, indicating that parakeets will utilize alternative cavities when preferred sites are not available, bringing them into greater conflict with other cavity nesters. Although some nuthatches remain in areas dominated by ring-necked parakeets, our results raise conservation concerns for the vulnerability of native cavity-nesting birds to the expansion of invasive parakeet populations. 相似文献
20.
Camilla WambergSøren Christensen I. JakobsenA.K. Müller S.J. Sørensen 《Soil biology & biochemistry》2003,35(10):1349-1357
Pea plants were grown in γ-irradiated soil in pots with and without addition of the AM fungus Glomus intraradices at sufficient N and limiting P. Depending on the growth phase of the plant presence of AM had negative or positive effect on rhizosphere activity. Before flowering during nutrient acquisition AM decreased rhizosphere respiration and number of protozoa but did not affect bacterial number suggesting top-down regulation of bacterial number by protozoan grazing. In contrast, during flowering and pod formation AM stimulated rhizosphere respiration and the negative effect on protozoa decreased. AM also affected the composition of the rhizosphere bacterial community as revealed from DNA analysis (DGGE). With or without mycorrhiza, rhizosphere respiration was P-limited on very young roots, not nutrient limited at more mature roots and C-limited at withering. This suggests changes in the rhizosphere community during plant growth also supported by changes in the bacteria (DGGE). 相似文献