稳定表达山羊SLAM受体的BHK-21细胞系的建立及应用

信号淋巴激活分子(SLAM)又称为CD150,是小反刍兽疫病毒(PPRV)和犬瘟热病毒(CDV)等麻疹病毒属病毒感染淋巴细胞的主要受体,在病毒侵入细胞中发挥重要作用。为建立稳定表达山羊SLAM(g SLAM)的真核细胞系,本研究将人工合成的g SLAM基因克隆至真核表达质粒p IRESpuro3中,并在该基因的3'端引入Flag标签序列作为分子标记,构建了重组质粒p IRES3-g SLAM。将该重组质粒转染BHK-21细胞,经嘌呤霉素加压筛选及采用表达绿色荧光蛋白(GFP)的重组PPRV病毒(r PPRV/GFP)感染鉴定后,筛选到稳定表达g SLAM基因的细胞系。r PPRV/GFP感染和western blot鉴定表明,无论是否有嘌呤霉素压力的存在,该细胞系在传代至第20代,仍能稳定表达g SLAM蛋白。由于g SLAM氨基酸序列与犬的同源性较高,以表达GFP重组CDV强毒株(r CDV/GFP)感染该细胞系,病毒可以感染且能形成明显的细胞病变,表明该细胞系可用于CDV强毒分离和致弱机制等相关研究。     

Vegetation dynamics in burnt heather-gorse shrublands under different grazing management with sheep and goats

Vegetation dynamics in previously burnt shrublands dominated by gorse (Ulex gallii) in north-west Spain were studied during 4 years under different grazing managements with sheep (Gallega breed) and goats (Cashmere or local breed). Initially (Period 1), treatments consisted of sheep or goat grazing on four plots (two replicates). After two grazing seasons, the plots were split in two and the treatments were reversed in four of the subplots; sheep were placed in subplots previously grazed by goats and vice versa (Period 2). The results from Period 1 showed that goats control the regrowth of gorse (from 19.6 to 14.2% of cover) more effectively than sheep (17.8 to 27.0% cover; P < 0.05), favouring the increase in herbaceous plants (from 21.5 to 42.1% of cover under goat grazing and from 22.1 to 26.6% under sheep grazing; P < 0.01). In Period 2, after changing treatments, there was a clear effect of the previous management (P < 0.01), conditioning the dynamics of the different plant components. Where the same animal species were maintained for 4 years, the differences between sheep or goat grazing on gorse and herbaceous plant dynamics increased (24.4% of gorse and 35.2% of herbaceous plant cover under goat grazing vs 43.8% of gorse cover and 19.1% of herbaceous plant cover under sheep grazing). Animal species change buffered the differences between sheep and goats during the second Period.     

小反刍兽疫病毒及山羊痘病毒N-ITR基因二联实时荧光定量RT-PCR检测方法的建立

Peste des petits ruminants virus (PPRV) and goat pox virus (GTPV) are the causative agents of two kinds of goats’ diseases-peste des petits ruminants and goat pox which can cause disaster economic losses. In order to detect the two viruses simultaneously and quickly, two sets of primers and relative probes were designed based on the nucleoprotein (N) gene of PPRV and the inverted terminal repeat (ITR) segment of GTPV， respectively. In order to work together in the same reaction, the probes were labeled with different fluorescent materials 5′FAM-TAMRA3′and 5′JOE-Eclipse3′，respectively. Results showed that the duplex Real-time RT-PCR assay was identified to be specific for PPRV and GTPV only and specific fluorescent signal could be detected, but the related viruses including fowl pox virus（FPV）and canine distemper virus (CDV) had no specific fluorescent signal. Positive recombinant plasmids (PPRV pMD18-T-N and GTPV pMD18-T-ITR) were built and used for positive quantitative templates to establish duplex standard curves. The developed assay based on the probe N-ITR was found to be highly specific and sensitive with a detection limit of 10² copies/μL cDNA and 10³ copies/μL DNA for PPRV and GTPV， respectively. Finally, the duplex Real-time RT-PCR assay for simultaneous detection of PPRV and GTPV was established preliminarily in the study.     

瘤胃液移植在反刍动物中的研究进展

瘤胃液移植是一种将健康供体动物的瘤胃液转移给受体动物的技术。目前，针对瘤胃液移植的报道还相对有限，但越来越多的研究结果显示，瘤胃液移植可以影响反刍动物的干物质采食量、产奶量等，还具有重建瘤胃菌群结构的潜力。本文综述了瘤胃液移植的方法及实践中的应用研究，分析其对反刍动物生长性能、瘤胃发酵、瘤胃微生物区系的影响以及在治疗某些疾病方面的作用，为反刍动物瘤胃液移植的研究提供参考。     

小反刍兽疫N5蛋白的原核表达及间接ELISA检测方法的建立

【目的】建立一种适用于PPRVN蛋白抗体的间接ELISA检测方法,应用于PPR防疫.【方法】根据GenBank中登录小反刍兽疫病毒(PPRV)Nigeria 75/1株N基因序列进行人工合成,设计1对特异引物对N5基因扩增,经测序分析后将N5基因片段和原核表达载体pET-32a(+)相连接,成功构建pET-32a-N5质粒.将pET-32a-N5转化于TransB(DE3)原核表达感受态细胞后用IPTG诱导表达获得重组蛋白,经SDS-PAGE分析和Western-blot鉴定,重组蛋白分析具有良好的反应原性.【结果】利用原核表达纯化的PPRV N重组蛋白作为包被抗原,初步建立了一种能够检测针对PPRV N蛋白抗体的间接ELISA方法.用建立的方法对112山羊份血清进行了抗体检测,该方法与竞争ELISA试剂盒对比,临床符合率94.8%.【结论】该方法具有较好的敏感性、重复性和特异性.     

Application of long‐chain alcohols as diet‐composition markers in sheep fed on grass–white clover and heather–gorse plant species

Utilization of long‐chain alcohols (LCOH) as diet‐composition markers in sheep consuming six diets composed of improved pasture species (Lolium perenne and Trifolium repens) with heather–gorse components (Erica spp., Calluna vulgaris and Ulex gallii) was evaluated. Twenty‐four adult cross‐bred sheep were housed in individual stalls. Diet composition was estimated from LCOH concentrations, combined or not with alkanes and long‐chain fatty acid (LCFA) data using least‐squares procedures. Prior to calculations, faecal concentrations were corrected using mean treatment (faecal recovery 1, FR1) recoveries and mean recoveries across diets (FR2). Estimates were compared with those obtained without faecal correction (FR0) and known values. Large differences between plant species and plant parts were found in LCOH patterns and total LCOH concentrations. LCOH provided complementary information to that given by alkanes and LCFA. Even‐chain LCOH comprised the largest fraction, representing on average 0·894 of total concentrations. Faecal recovery was incomplete and tended to increase with carbon‐chain length (CCL) in a curvilinear manner (FR = ?7·872 + 0·580 × CCL ? 0·010 × CCL²; P < 0·001; r² = 0·752). Diet composition influenced (P < 0·001) LCOH recovery, although variability within (CV of 9·0%) and between (CV of 13·3%) diets was low. Accuracy of estimates was influenced (P < 0·001) by faecal‐correction method and markers used, and the best estimate was obtained combining LCOH and alkanes. Improvement in estimate accuracy can be achieved if suitable correction of LCOH faecal concentrations is performed prior to calculations. Results indicated that LCOH showed lower dependence than alkanes and LCFA on the use of accurate faecal‐correction data.     

向日葵副产物的营养特性及在反刍动物中的应用

饲料资源缺乏一直以来都是制约中国畜牧业发展的主要因素。从长远看，常规饲料已不能维持中国畜牧业的可持续发展，开发新的饲料资源是解决饲料不足的重要途径之一。向日葵副产物营养价值较高、来源广、价格低，是一种非常具有开发价值的新型饲料。向日葵副产物包括向日葵饼粕、向日葵盘、向日葵秸秆及葵花籽壳：向日葵饼粕蛋白质含量高、纤维含量低，是一种优质的植物蛋白来源，可以提高奶牛乳汁的营养价值、促进肉牛生长、提高羊的瘤胃发酵效果；向日葵盘粗脂肪含量高，含有大量的膳食纤维，可以提高奶牛的产奶量和乳脂率，促进肉牛增重，提高羊瘤胃挥发性脂肪酸浓度以及粗蛋白质、粗纤维、中性洗涤纤维的消化率；向日葵秸秆富含氮、磷、钾、钙、镁等矿物质元素以及少量的蛋白质，可以为肉牛提供一些常量元素和微量元素，提高羊的日增重，降低料重比；葵花籽壳的主要成分是纤维素和木质素，含有丰富的生物活性物质，可以提高奶牛日增重、肉牛饲料转化率。作者具体介绍了向日葵副产物饼粕、葵盘、秸秆、葵花籽壳的营养价值，以及目前它们在反刍动物饲料中的应用情况，以期为今后向日葵副产物的开发利用提供理论依据。     

小反刍兽疫病毒F蛋白抗原位点的克隆与表达

目的表达小反刍兽疫F蛋白的抗原位点用于检测与预防。方法用DNAStar分析小反刍兽疫的抗原位点,采用逆转录聚合酶链式反应(RT-PCR)技术,从病羊组织中PPRV的F基因的抗原位点并克隆到原核表达载体PET-28b中,最后用PCR、酶切和测序分析对重组质粒进行鉴定;将重组质粒转化到大肠杆菌Rosetta中,经ITPG诱导表达PPRVF基因的抗原位点;用SDS-PAGE和Western-Blotting分析抗原位点蛋白的表达。结果将RT-PCR产物电泳,得到与预期大小相符的特异性片段;对重组质粒PET-28b-F35-111(pZLW013)、PET-28b-F143-485(pZLW014)和PET-28b-F323-485(pZLW015)酶切后,均出现与预期相符的片段;DNA测序表明插入的片段的序列与小反刍兽疫F基因的抗原位点序列分别完全一致,其大小分别为251bp、1053bp和514bp;将重组表达的蛋白经SDS-PAGE和West-ern-Blotting分析,证明小反刍兽疫抗原位点F35-111没有表达、抗原位点F143-485和F323-485得到表达。结论成功表达了PPRVF基因的两段抗原位点蛋白,为日后检测与预防工作奠定了基础。     

小反刍兽疫病毒H蛋白原核表达的研究

小反刍兽疫病毒属副粘病毒科麻疹病毒属,为一不分节段的负链RNA病毒。本研究以灭活的检测用抗原为材料,抽提基因组RNA作为模板,根据GenBank下栽的序列及载体的需要设计其主要的抗原基因H基因的编码序列的RT—PCIL扩增引物;然后将扩增基因片段与T载体连接克隆测序,再与原核表达载体pET100／D—TOPO连接、诱导表达。结果显示所设计引物对模极有预期扩增,序列测定表明为正确片段;片段经纯化与表达载体连接,已挑到阳性克隆。H基因的表达菌经IPTG诱导,SDS—PAGE电泳。结果显示：H基因有预期分子量大小的蛋白表达;用Anti—His抗体已检测到相应融合蛋白的表达,其原核表达的相应蛋白抗原性状况正在试验研究中,表达产物具有潜在的诊断抗原和免疫原价值。