稳定表达猪繁殖与呼吸综合征病毒受体CD163的HEK293细胞系的建立

为构建稳定表达猪繁殖与呼吸综合征病毒(PRRSV)受体CD163的HEK293细胞系,本研究采用RT-PCR方法从猪肺泡巨噬细胞(PAM)总RNA中扩增CD163基因并将其克隆至真核表达载体pc DNA3.1(+)中,构建重组质粒pc DNA-CD163。将该重组质粒转染HEK293细胞,并利用G418筛选、纯化,获得了稳定表达CD163受体蛋白的HEK293CD163细胞系。RT-PCR、流式细胞术及western blot检测结果表明,该细胞系在传代至15代后,CD163在HEK293细胞中仍然能够稳定表达。在该细胞系中接种PRRSV后进行间接免疫荧光及噬斑试验检测表明,病毒能够通过CD163感染HEK293CD163细胞并在其中增殖。该细胞系的建立为进一步研究PRRSV的侵入细胞和致病机制提供了病毒在体外增殖的易感细胞系。     

猪繁殖与呼吸综合征病毒RdRp基因的克隆与原核表达

将猪繁殖与呼吸综合征病毒RNA依赖性的RNA聚合酶(RdRp)基因进行克隆、表达,并获得纯化重组蛋白,为进一步研究猪繁殖与呼吸综合征病毒RdRp的功能奠定基础.以RdRp基因cDNA为模板,用PCR扩增出RdRp基因片段,应用定向克隆策略将扩增后片段与高效表达载体pET32a(+)重组,阳性克隆重组质粒经酶切及测序鉴定,IPTG诱导高效表达.PCR法扩增出720 bp的片段,酶切及测序鉴定获得正确的重组质粒,在大肠埃希菌LB21中表达得到分子质量约为46 ku的融合蛋白.成功克隆及表达了猪繁殖与呼吸综合征病毒RdRp基因.     

PRRSV清道夫受体CD163基因的真核表达和功能鉴定

从猪肺泡巨噬细胞(PAM)中提取总RNA为模板,采用RT-PCR法获得猪繁殖与呼吸综合征病毒(PRRSV)的受体CD163全基因,将该基因克隆到真核表达载体pcDNA3.1/V5-HIS A上,构建出真核重组质粒pcDNA3.1-CD163,经酶切和DNA测序证明获得了CD163基因,其序列与GenBank报道序列比较,核苷酸同源性为99.37%。将测序正确的CD163基因在脂质体LipofectamineTM2000介导下转染PK-15细胞,通过间接免疫荧光(IFA)检测到了CD163在PK-15中的表达。将转染的细胞感染PRRSV后,经IFA检测转染CD163的细胞能够被PRRSV感染。     

猪繁殖与呼吸综合征病毒感染相关受体的研究概况

猪繁殖与呼吸综合征病毒感染细胞主要是通过敏感细胞上特异性受体介导产生的。文章对猪繁殖与呼吸综合征病毒感染相关受体的研究进行了综述,硫酸乙酰肝素、唾液酸黏附素和CD163是主要的相关受体,对它们的功能特性研究将为猪繁殖与呼吸综合征病毒的致病机理和预防研究提供理论支持。     

猪繁殖与呼吸综合征RT-PCR诊断及ORF5基因原核表达载体的构建

参照GanBank上发表的猪繁殖与呼吸综合征病毒核苷酸序列,利用生物软件,设计1对ORF5基因的特异性引物,用Trizol法提取病料的总RNA,进行RT-PcR扩增后获得目的基因,将其连接到PMD18-T载体后转入大肠杆菌Top10细胞中,抽提质粒并双酶切,回收目的基因,然后克隆于原核表达载体PET-32a(+)中,通过抗性筛选、酶切鉴定和序列测定,证实得到含有目的基因的阳性克隆,本实验成功构建了猪繁殖与呼吸综合征病毒贵州分离株ORF5基因原核表达载体PET-ORF5,为进一步研究ORF5基因的原核表达及猪繁殖与呼吸综合征的基因工程疫苗的研究奠定了基础.     

稳定表达猪CD163受体的MARC-145细胞系的构建

为了构建稳定表达猪CD163(pCD163)受体的MARC-145细胞系,从猪肺泡巨噬细胞(PAMs)中提取总RNA,通过RT-PCR方法扩增pCD163基因,将pCD163基因克隆到慢病毒载体pLVX-IRES-mCherry中,获得重组质粒pLVX-pCD163-IRES-mCherry。将重组质粒pLVX-pCD163-IRES-mCherry与辅助质粒psPAX2、VSVG共转染293T细胞,获得具有感染能力的慢病毒粒子,使用慢病毒感染MARC-145细胞,用嘌呤霉素初步筛选阳性细胞,采用终点稀释法筛选出稳定表达pCD163受体的MARC-145细胞。通过RT-PCR扩增pCD163基因及测序分析表明细胞系基因组中存在pCD163受体编码序列,IFA和Western blot试验验证了pCD163受体蛋白在细胞系中稳定表达。用不同谱系的猪繁殖与呼吸综合征病毒(PRRSV)GXNN1396(lineage 8)、GXNN202004a(lineage 1)、GXGG202007(lineage 3)分别感染稳定表达pCD163的MARC-145细胞系与MARC-145细胞,PRR...     

表达猪繁殖与呼吸综合征病毒CH-1a株核衣壳蛋白重组鸡痘病毒的构建

从含有猪繁殖与呼吸综合征病毒（PRRSV）核衣壳蛋白（N）的质粒扩增出N基因，构建禽痘病毒转移载体。该载体含有禽痘病毒早晚期启动子LP2EP2控制之下的PRRSVN基因、P11启动下的报告基因lacZ以及用于同源重组的禽痘病毒基因组的片段。在转移载体转染亲本病毒S—FPV-017感染的鸡胚成纤维细胞（CEF）之后，采用蓝色表型筛选的方法，筛选到表达N基因的重组病毒，并对其进行了6轮蚀斑纯化。PCR方法鉴定证明重组病毒的基因组中含有完整PRRSVN基因，间接免疫荧光试验证明了PRRSVN蛋白在重组病毒感染的CEF细胞中获得表达，本研究为猪繁殖与呼吸综合征非复制型疫苗的研制打下了基础。     

猪繁殖与呼吸综合征病毒XH株感染性克隆的构建

为构建猪繁殖与呼吸综合征病毒(PRRSV)反向遗传操作系统,本实验采用RT-PCR方法将PRRSV-XH株的基因组序列分为6个片段进行扩增,连接至低拷贝载体pOKQ中,获得全长cDNA克隆。将该克隆体外转录,经脂质体转染Marc-145细胞,观察细胞病变。经RT-PCR、间接免疫荧光和western blot等试验验证,表明构建了具有感染性的PRRSV-XH株全长cDNA克隆。本实验为在分子水平上研究PRRSV的复制、致病机理和基因产物的功能等方面提供技术支持,并为构建新型病毒载体和开发安全有效的活病毒疫苗奠定基础。     

猪繁殖与呼吸综合征病毒细胞受体研究进展

猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)主要是通过与宿主细胞表面的特异性受体结合,利用细胞的内吞作用而感染易感细胞。作者对猪繁殖与呼吸综合征病毒相关受体的研究进行了综述,迄今已报道了5种独立的但功能相关的受体:硫酸乙酰肝素、唾液酸黏附素、波形蛋白、CD163和非肌肉肌动蛋白ⅡA。对受体的功能特性进行研究,将为PRRSV的感染机理和病毒性疾病的预防和治疗具有重要的意义。     

猪繁殖与呼吸综合征病毒贵州分离株ORF5基因的克隆及其表达载体的构建

为了研究猪繁殖与呼吸综合征病毒贵州分离株ORF5基因的表达,研究参照GenBank上发表的猪繁殖与呼吸综合征病毒贵州分离株(PRRSV Guizhou-1)的核苷酸序列,利用DNASTAR、Oligo6.0和Primer Premier 5.0软件设计1对ORF5基因的特异性引物,同时将贵州大学动物科学学院分子生物学实验室分离、保存的PRRSV Guizhou-1接种于Marc-145细胞,产生明显细胞病变后收集病毒,用Trizol法提取总RNA进行RT-PCR扩增后获得目的基因,并将其克隆于质粒pMD18-T载体后转入大肠杆菌Top10细胞中,然后分别克隆于原核表达载体pET-32a(+)和真核表达载体pcD-NA3.1(+)中,再进行抗性筛选、酶切鉴定和序列测定.结果表明:得到含有目的基因的阳性克隆,成功构建了猪繁殖与呼吸综合征病毒贵州分离株ORF5基因原核表达载体pET-ORF5及真核表达载体pcDNA-ORF5.     

商会自治的基石——商会自治规范研究

在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。     

癸氧喹酯干混悬剂含量测定的改进

采用高效液相色谱法测定癸氧喹酯干混悬剂的含量,在2-250μg/mL范围内,峰面积的常用对数与进样量浓度的常用对数呈良好的线性关系,R^2=1(n=5),平均回收率为99.24%~99.51%,RSD在0.05%~0.28%。此方法分析时间短,样品前处理简便、定量结果准确,重现性好,结果满意,为其质量控制提供了依据。     

猪毛色遗传的研究进展

本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。     

Comparison of 2 methods of centesis of the bursa of the biceps brachii tendon of horses

REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air.     

不同样本商品蜂蜜中硒含量的测定

用硝酸和高氯酸消化蜂蜜,使硒游离出来,在微酸性环境下,硒和2,3-二氨基萘(DAN)生成有较强荧光的物质,用环己烷萃取,在激发波长378nm,荧光波长518nm处测定其荧光强度。蜂蜜中硒含量范围:0.10～0.82μg/g。表明:蜂蜜应视为天然富硒营养品。     

乳酸杆菌益生作用机制的研究进展

乳酸杆菌作为益生菌广泛用于人和动物。本文综述了乳酸杆菌改善宿主健康的机制。乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道。文章重点讨论了乳酸杆菌表面成分（表面蛋白、脂磷壁酸和肽聚糖）与肠道受体（Ｃ型凝集素受体、Ｔｏｌｌ样受体和 Ｎｏｄ样受体）,阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制。     

Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF

Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo.     

Mechanism of Action of Probiotic Function of Lactobacilli

乳酸杆菌作为益生菌广泛用于人和动物.本文综述了乳酸杆菌改善宿主健康的机制.乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道.文章重点讨论了乳酸杆菌表面成分(表面蛋白、脂磷壁酸和肽聚糖)与肠道受体(C型凝集素受体、Toll样受体和Nod样受体),阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制.     

Evaluation of serum concentrations of biomarkers of skeletal metabolism and results of radiography as indicators of severity of osteochondrosis in foals

OBJECTIVE: To determine whether serum concentrations of biomarkers of skeletal metabolism can, in conjunction with radiographic evaluation, indicate severity of osteochondrosis in developing horses. ANIMALS: 43 Dutch Warmblood foals with varying severity of osteochondrosis. PROCEDURE: 24 foals were monitored for 5 months and 19 foals were monitored for 11 months. Monthly radiographs of femoropatellar-femorotibial and tibio-tarsal joints were graded for osteochondral abnormalities. Serial blood samples were assayed for 8 cartilage and bone biomarkers. At the end of the monitoring period, foals were examined for macroscopic osteochondrosis lesions. RESULTS: Temporal relationships were evident between certain serum biomarkers and osteochondrosis severity in foals during their first year. Biomarkers of collagen degradation (collagenase-generated neoepitopes of type-II collagen fragments, type-I and -II collagen fragments [COL2-3/4C(short)], and cross-linked telopeptide fragments of type-I collagen) and bone mineralization (osteocalcin) were positive indicators of osteochondrosis severity at 5 months of age. In foals with lesions at 11 months of age, osteochondrosis severity correlated negatively with COL2-3/4C(short) and osteocalcin and positively with C-propeptide of type-II procollagen (CPII), a collagen synthesis marker. Radiographic grading of osteochondrosis lesions significantly correlated with macroscopic osteochondrosis severity score at both ages and was strongest when combined with osteocalcin at 5 months and CPII at 11 months. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of serum biomarkers to indicate osteochondrosis severity appears to depend on stage of disease and is strengthened with radiography. In older foals with more permanent lesions, osteochondrosis severity is significantly related to biomarker concentrations of decreased bone formation and increased cartilage synthesis.