牛源犬新孢子虫NcSRS2-NcGRA7融合基因的克隆与生物信息学分析

为了解牛源犬新孢子虫NcSRS2-NcGRA7融合基因的生物学特性,本试验提取牛源犬新孢子虫基因组DNA,应用PCR技术扩增犬新孢子虫表面蛋白基因NcSRS2和致密颗粒蛋白基因NcGRA7,SOE-PCR技术拼接NcSRS2和NcGRA7基因,构建NcSRS2-NcGRA7融合基因重组克隆质粒,并进行PCR鉴定、双酶切鉴定及生物信息学分析。结果,NcSRS2基因扩增片段大小为1 061 bp,NcGRA7基因扩增片段大小为364 bp,NcSRS2-NcGRA7融合基因扩增片段大小为1 482 bp;获得重组克隆质粒pMD18-NcSRS2-NcGRA7经PCR鉴定、双酶切鉴定正确;测序分析表明,与Gen-Bank中已发表的美国株犬新孢子虫(AF061249、AF176649)核苷酸序列同源性为99%;经DNAman等软件分析,预测NcSRS2-NcGRA7融合蛋白抗原指数较高,融合蛋白二级结构以α-螺旋和β-折叠为主,三级结构中2种蛋白独立折叠,并借助Linker互相连接,功能互不影响。本试验为牛源犬新孢子虫NcSRS2-NcGRA7融合蛋白的免疫学研究奠定了基础。     

提高贯通培养项目基础文化课教学质量的策略研究

高端技术技能人才贯通培养实验项目(简称贯通培养项目)是北京市为更好地适应国际化产业发展,培养高素质技能型人才所推行的新型教学模式,涵盖了高中教育、职业教育、本科教育阶段。通过对基础文化课阶段的教学质量现状进行调查分析发现,这一项目在人才培养方案、课程体系、教学督导、师资队伍等方面获得一定经验,也存在一定问题。要提高基础文化课阶段的教学质量,需要在上述四个方面进一步改进。     

覆膜和密度对‘陇薯7号’产量及不同质量块茎分布的影响

以‘陇薯7号’原种为试验材料,采用裂区设计,研究覆膜和密度对其成熟期、产量及其构成因素以及不同质量块茎分布的影响,综合分析产量、成本、效益,提出高寒阴湿区‘陇薯7号’繁种适宜的种植方式和合理的种植密度,为其北繁南种脱毒种薯繁育技术体系的形成提供理论支持,以及在南方冬作区的大面积推广奠定基础。结果表明:黑色地膜覆盖能使‘陇薯7号’成熟期提前15 d,生育期缩短11 d,产量增加57.19%。随着密度的增加,有效株数增加,单株结薯数减少,单薯质量降低,产量显著增加。在试验设计的密度范围内,产量与密度呈线性正相关,在最高密度下获得最高产量。随着密度的增加,20 g、20～50 g块茎比率呈直线上升趋势,100g块茎比率呈直线下降趋势, 50～100 g块茎比率因种植方式不同而变化不同,地膜覆盖种植,呈先升后降的趋势,密度13.5×10~4 hm~(-2)时达到最高,露地种植则表现为持续下降的趋势。地膜覆盖主要影响20～50 g和100 g块茎比率。综合分析产量、成本和效益,以地膜覆盖密度增加到13.5×10~4 hm~(-2)为试验最佳处理,产量48 938.89 kg/hm~2,纯收益1673.53元/hm~2,20～50 g块茎比率27.15%,50～100 g块茎比率36.08%。建议在高寒阴湿区繁育‘陇薯7号’脱毒一级种采用黑色地膜覆盖种植,密度13.5×10~4 hm~(-2),块茎按质量大小进行分级。     

浅谈“7S”法在高校实验室安全管理中的应用

高校实验室是人才培养、科学研究、文化传承的重要基地。近年来,高校实验室数量日益增加,并且逐渐全面开放,面对大量的学生和教师,实验室安全事故时有发生,这些问题关系到高校以及社会的安全和稳定,故提高实验室管理势在必行。本文对近些年高校实验室安全事故的类型及发生原因进行了概括,并总结了目前高校实验室普遍存在的问题,阐述了"7S"法管理的基本内容,提出用"7S"法管理理念来加强实验室的安全管理。     

苏云金芽胞杆菌杀虫晶体蛋白Cry7Ba1溶解性分子改良

苏云金芽胞杆菌（Bacillus thuringiensis）杀虫晶体蛋白Cry7Ba1需要在强碱性缓冲液中（pH12.5）才能溶解,而且只有溶解后才能对小菜蛾（Plutella xvlostella）表现出毒力,其LC50为1.33μg/mL。分别用Cry1Ac和Cry1C晶体蛋白的C-半段替换Cry7Ba1的C-半段,结果发现当Cry7Ba1的C-半段被替换后,重组晶体蛋白与Cry1Ac和Cry1C等其它晶体蛋白一样在弱碱性条件下（pH9.5）能有效溶解,而且重组晶体蛋白对小菜蛾的杀虫活性没有发生明显变化,其LC50分别为1.32和1.39μg/mL。结果还显示重组晶体蛋白不需要经过体外溶解就能对小菜蛾表现出杀虫活性。研究结果揭示Cry7Ba1晶体蛋白难溶的特性是由其C-半段结构决定的,通过改变其C-半段结构改善溶解性,为该晶体蛋白在害虫生物防治中的应用提供了可能。     

H7 亚型禽流感病毒 HA 蛋白在Sf9 中的表达与纯化

【目的】真核表达 H7 亚型禽流感病毒（Avian influenza virus, AIV）HA 蛋白,为进一步制备 H7 亚型 HIV 亚单位疫苗,评价其免疫原性提供基础。【方法】首先根据草地贪夜蛾卵巢细胞（Spodoptera frugiperda clone 9, Sf9）的密码子偏嗜性,优化并合成 H7 亚型 AIV 的 HA 序列,将其克隆至穿梭质粒 pFastBac1 中;接着将重组HA基因的杆状病毒质粒转染至 Sf9 中,通过间接免疫荧光和 Western blots 试验鉴定重组病毒是否拯救成功;再通过 SYBR green 染料法荧光定量 PCR 试验,建立基于 gp64 基因的杆状病毒 qPCR 定量方法,筛选出在 Sf9 中表达 HA 重组蛋白的最佳感染复数和孵育时间;最后通过 His 镍株亲和纯化 HA 重组蛋白。【结果】表达 H7 亚型 AIV HA 蛋白的重组杆状病毒在 Sf9 盲传 3 代后,Sf9 开始出现肿胀、脱落、死亡等细胞病变,表明重组杆状病毒拯救成功;通过间接免疫荧光和 Western blots 试验发现,Sf9 内出现可与 HA 重组蛋白特异性结合的绿色荧光信号,特异性结合的 HA 重组蛋白条带大小约 70 kD 左右,与预期相符,且 HA 重组蛋白可与 H7 亚型 AIV 阳性血清反应,表明 HA 重组蛋白表达成功;以构建的 pUC19-gp64 质粒为标准,建立基于 gp64 基因的杆状病毒qPCR 检测方法,该方法在 1×103~1×108 copy/μL 间呈现良好的线性关系,标准曲线的相关系数 R2=0.993;利用qPCR 检测方法对杆状病毒液滴度进行定量,并通过 Western blots 试验筛选 HA 蛋白表达的最佳感染复数和孵育时间,结果显示以 10 MOI 的比例接种 Sf9,孵育 72 h,蛋白表达效果最好;通过 His 镍株亲和纯化 HA 重组蛋白,蛋白浓度为 0.268 mg/mL,鸡红细胞凝集效价为 4 log2。【结论】本试验在 Sf9 中成功表达并纯化 H7 亚型 AIV HA 蛋白,并建立与之相应的杆状病毒荧光定量 PCR 检测方法,可为进一步评价 H7 亚单位疫苗的免疫原性提供参考。     

G and P genotype profiles of rotavirus A field strains circulating in beef and dairy cattle herds in Brazil, 2006–2015

The aim of this retrospective study was to use RT-PCR and nucleotide sequencing analysis to determine the G (VP7 gene) and P (VP4 gene) genotypes of 155 Brazilian bovine rotavirus A (RVA) wild-type strains detected in diarrheic calves from all Brazilian geographical regions from 2006 to 2015. The RVA strains evaluated belonged to the G6, G10, P[5], and P[11] genotypes. The G6P[5] genotype was prevalent (65.5%; P < 0.05) in beef, and the G10P[11] (38.4%) and G6P[11] (30.8%) genotypes were more prevalent in dairy cattle herds. The Midwest was the region with the highest number of genotyped RVA strains, where the genotypes G6, P[5], and P[11] were identified. Genotype combination G6-IV/P[5]-IX, prevalent in beef herds, and G6-III/P[11]-III or G10-IV/P[11]-III, prevalent in dairy herds, were detected. In addition, for the first time in Brazil, we detected the P[5] and P[11] genotype RVA strains that belong to lineage II and VII, respectively.     

不同营养水平日粮对陕北白绒山羊生长性能和小肠组织SLC7A7、SLC3A1及SLC15A1 mRNA表达的影响

本试验旨在研究日粮营养水平对断奶后2~6月龄陕北白绒山羊生长性能及小肠组织中与氨基酸转运吸收相关的SLC7A7、SLC3A1和SLC15A1 mRNA表达的影响。选取健康、日龄（（60±1.60）d）和体重（（10.73±1.03）kg）相近的雌性陕北白绒山羊羔羊36只，随机分为4组，分别饲喂4种试验日粮，其消化能和粗蛋白质水平分别为标准日粮的85%、100%、115%和130%。标准日粮营养水平参考肉羊饲养标准NY/T816-2004，依据生长阶段（10~19 kg、15~27 kg）和目标增重设置。试验期间，分别于120和180日龄称重，于180日龄，每个重复屠宰1只试验羊，采集十二指肠中上部、空肠中段、回肠末端组织样品，通过实时荧光定量PCR检测SLC7A7、SLC3A1和SLC15A1表达水平。结果表明：1）第一阶段（60~120 d）115%水平组平均日增重显著高于其他三组（P<0.05），第二阶段（121~180 d）115%水平组平均日增重显著高于85%和100%水平组（P<0.05），与130%水平组无明显差异。两阶段115%水平组羔羊干物质采食量极显著高于其他3组（P<0.01）。两阶段料重比115%水平组显著低于85%、100%水平组（P<0.05）。2）相同营养水平下，SLC7A7和SLC15A1 mRNA的表达丰度顺序均为回肠>空肠>十二指肠；3）随日粮营养水平的增加，SLC7A7、SLC3A1和SLC15A1 mRNA在小肠各段的相对表达量呈先上升后下降的趋势，且115%水平组表达量最高。115%水平组SLC7A7和SLC15A1 mRNA相对表达量显著高于其他3组（P<0.05）；115%水平组SLC3A1 mRNA的相对表达量显著高于85%和130%水平组组（P<0.05）。本试验条件下，与其他营养水平组相比，115%水平组陕北白绒山羊羔羊在2~6月龄生长性能最佳，SLC7A7、SLC3A1和SLC15A1 mRNA在小肠各段的表达量最高。     

Abomasal infusion of casein, starch and soybean oil differentially affect plasma concentrations of gut peptides and feed intake in lactating dairy cows

The effects of specific nutrients on secretion and plasma concentrations of gut peptides (glucagon-like peptide-1((7-36)) amide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin-8 (CCK)) differ across species, but are not reported for cattle. Our objective was to determine acute (hours) and chronic (1 week) effects of increased abomasal supply of protein, carbohydrate, or fat to the small intestine on dry matter intake (DMI) and plasma concentrations of GLP-1, GIP, CCK, and insulin. Four mid-lactation Holstein cows were used in a 4 x 4 Latin square design experiment. Treatments were 7-day abomasal infusions of water, soybean oil (500 g/d), corn starch (1100 g/d), or casein (800 g/d). Jugular vein plasma was obtained over 7h at the end of the first and last day of infusions. Oil infusion decreased DMI on day 7, but total metabolizable energy (ME) supply (diet plus infusate) did not differ from water infusion. Casein and starch infusion had no effect on feed DMI; thus, ME supply increased. Decreased DMI on day 7 of oil infusion was accompanied by increased plasma GLP-1 concentration, but decreased plasma CCK concentration. Increased plasma GIP concentration was associated with increased ME supply on day 7 of casein and starch infusion. Casein infusion tended to increase plasma CCK concentration on both days of sampling, and increased plasma GLP-1 and insulin concentration on day 1 of infusion. The present data indicate a sustained elevation of plasma concentration of GLP-1, but not CCK, may contribute to the reduced DMI observed in dairy cows provided supplemental fat.     

Avian influenza outbreak management: action at time of confirmation, depopulation and disposal methods; the 'Belgian experience' during the H7N7 highly pathogenic avian influenza epidemic in 2003

Eradication of H5 and H7 influenza in a positive flock will include mass depopulation of birds, containment and inactivation of the virus in the carcasses and litter, and decontamination of the facility. A quick response is desired in the event of a disease outbreak. Ideally, birds should be depopulated within 24 h after detecting the virus. Mass depopulation of birds must be performed in a humane manner while minimizing human health and biosecurity risks. In the framework of the European legislation, a number of methods are authorized for the killing of poultry for processing prior to marketing. However, during emergencies such as a disease outbreak, there are fewer options. The current most commonly used procedures for large-scale emergency depopulation of birds consist of exposing poultry to CO or CO(2) gas. Both gasses have been used in Belgium during the H7N7 crisis in 2003. The gassing procedures include whole house gassing, portable panel enclosures, cage cabinets, containers and polyethylene tent method. Whole house gassing requires sealing the house to prevent gas leakage and, using specialized equipment, introducing large volumes of gas evenly over the birds. All procedures are very labour intensive, create a biosecurity risk and require a large number of personnel. There are considerable region-to-region differences in emergency depopulation techniques and disposal of carcasses and infected material. Because of the differences in bird type and species, management, housing and stocking density, it is difficult to propose a depopulation technique that will be suitable for all circumstances. Safety of the human operators is an increasing concern with all H5 and H7 strains and in particular with the highly pathogenic H5N1 strain. Researchers and commercial poultry companies in the United States recently established that non-toxic water-based foam with a certain bubble size presents a practicable, effective and humane method for mass depopulation. Foam of the right bubble size creates an occlusion in the trachea of birds, causing a rapid onset of hypoxia. The foam that blankets the broiler house induces physical hypoxia - the same cause of death as the approved method using carbon dioxide gas (CO(2)). The article illustrates the different culling and disposal methods with a focus on the methods used during the 2003 H7N7 crisis in Belgium.