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。     

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.     

中国西藏小反刍兽疫的发生状况与防控

2007年7月,我国首次在西藏自治区阿里地区发生了小反刍兽疫疫病流行.从2007年7月9日到11月15日,先后在阿里地区革吉、日土、札达、改则4个县出现疫情,共20个疫点,羊发病6122只,死亡1888只.2008年6月,在那曲地区尼玛县双湖区再次发现小反刍兽疫疫情,病羊及同群羊合计6690只.为消灭该病,采取了扑杀、...     

苜蓿植株再生及PPRV-F基因转化条件的研究

以苜蓿品种甘农1号下胚轴为外植体,研究不同培养基对苜蓿愈伤组织、体细胞胚形成的影响,通过体细胞胚发生途径建立再生体系,在此基础上,摸索农杆菌介导小反刍兽疫病毒F基因的转化条件。结果表明:所选培养基愈伤组织诱导率均为100%,UM+0.1mg/L NAA+0.5mg/L KT为诱导体细胞胚的最佳培养基。农杆菌介导的苜蓿遗传转化条件为:下胚轴预培养3d,农杆菌菌液侵染15min,然后在培养基上铺一层灭菌滤纸共培养3d后清洗,选择压为卡那霉素(Km)75mg/L,抑菌浓度为头孢霉素(Cef)300mg/L。本研究为制备防治小反刍兽疫转基因植物疫苗奠定了基础。     

反刍动物木本饲料的组合效应

本文综述了反刍动物木本饲料的组合效应,并对该类饲料(多含次生代谢物)的组合效应进行了分类,指出了其与常规饲料组合效应的异同及今后值得重点研究的几个问题。同时还就此类饲料组合效应的研究方法进行了比较,指出应在体外产气技术的基础上结合动物饲养试验来进行木本饲料的组合效应及其营养代谢机理的研究。     

小反刍兽疫诊断及其免疫防制的研究进展

小反刍兽疫(peste des petits ruminants,PPR)是由副黏病毒科麻疹病毒属小反刍兽疫病毒(peste des petits ruminants virus,PPRV)引起的一种急性、烈性、接触性传染病,目前已给中国养羊业造成巨大损失与威胁。作者对PPR的流行病学、诊断方法及疫苗研制进展等方面作一简要综述,以期为该病的防控提供有效的参考。     

Metabolic characteristics in ruminants of the proteins in newly developed hull-less barley varieties with altered starch traits

Recently, new varieties of hull-less barley have been developed with altered carbohydrate traits. To our knowledge, there is no study on metabolic characteristics in ruminants of the proteins in the newly developed hull-less barley varieties. The objectives of this study were to: compare metabolic characteristics of the proteins of zero-amylose waxy (CDC Fibar), low-amylose waxy (CDC Rattan), high-amylose (HB08302), and normal starch (CDC McGwire) hull-less barley. In situ animal trials were carried out to generate the original rumen fermentation data for modeling nutrient supply to dairy cattle by using two dairy nutrition models – Dutch DVE/OEB system and NRC 2001. The major comparisons were made in terms of i) truly absorbed protein in the small intestine (DVE or MP, and ii) degraded protein balance (DPB). The study revealed that zero-amylose waxy hull-less barley was superior (P < 0.05) in both DVE (123 vs. 117, 114, 103 g kg⁻¹ DM) and MP (112 vs. 93, 96, and 87 g kg⁻¹ DM) when hull-less barley was evaluated as a single feed for dairy cattle. All of four hull-less barley varieties had negative DPB (DPB^OEB; −37.4, −17.1, −30.2, and −28.2 g kg⁻¹ DM for normal starch, zero-amylose waxy, waxy, and high-amylose cultivar, respectively), indicating the potential N shortage. In conclusion, the alteration of starch structure in granule provided a relatively balanced energy and protein for microbial synthesis in the rumen. The DVE and DPB predicted by using the DVE/OEB system can be explained (r² > 0.76) by the equivalent parameters, predicted by using the NRC 2001 model. The alteration of starch structure in granule affects metabolic characteristics of the proteins of hull-less barley in ruminants.     

喷雾-复凝聚法制备小反刍兽疫缓释微囊疫苗的研究

建立了喷雾-复凝聚法制备灭活PPR微囊疫苗的工艺方法,并对制备的疫苗进行了主要性能指标测定和动物试验。结果显示：三批微囊形态均呈不规则长囊状,平均包埋率为42%,包埋前后灭活PPRV抗原活性得到较好保持。制备的灭活PPR微囊疫苗在免疫绵羊后1个月,中和抗体滴度均能达到1∶20以上,均高于小反刍兽疫弱毒疫苗1∶10的质量标准,最高可达到1∶160;且中和抗体滴度至少能持续7个月,呈现出良好的缓释效果,是一种具有广阔应用前景的新型疫苗。