猪传染性胸膜肺炎放线杆菌ApxⅣ毒素基因的克隆与表达

以猪传染性胸膜肺炎放线杆菌（APP）血清2型标准菌株基因组DNA为模板,用PCR方法扩增ApxⅣ毒素基因特异片段1.5 kb左右,将PCR产物纯化后与pMD18-T连接并测序,结果该片段的碱基序列与GenBank中标准株序列的同源性为98%。随后将该片段亚克隆到原核表达载体pET-28a（＋）的多克隆位点,经鉴定后得到重组质粒pET-ApxⅣ,将此重组质粒转化到受体菌BL21-DL3中,并用诱导剂乳糖进行诱导表达,5 h后表达达到高峰。经12%SDS-PAGE电泳检测,表达得到的融合蛋白约为61 000。经Western blotting分析,表达蛋白能与APP阳性血清发生特异性反应,而与阴性血清不反应。     

猪传染性胸膜肺炎放线杆菌外膜蛋白基因的克隆和表达

以猪传染性胸膜肺炎放线杆菌（APP）血清7型25-4株基因组DNA为模板，用PCR扩增外膜蛋白（OMP）基因特异片段，并克隆于pMD18-T中，经酶切及核苷酸序列分析鉴定后，亚克隆于原核表达栽体pGEX-6P-1，成功构建了重组表达载体pGEX-omp；以此转化大肠埃希氏菌BL21（DE3），经SDS-PAGE鉴定，表达的可溶性融合蛋白分子质量约为61 ku，命名为GST-OMP。以GST亲和层析柱纯化并利用Xa因子酶解，获得切掉标签的OMP。经ELISA检测，该OMP蛋白能够与兔抗APP的阳性血清反应，具有很好的免疫活性。GST-OMP蛋白的成功表达为APP OMP相关分子生物学功能的研制奠定了基础。     

鸡组织细胞端粒相关序列的克隆及序列分析

根据真核细胞端粒DNA序列的共同特点，设计了一条引物（TELO02）（24nt)。按照TaKala克隆试剂盒的介绍方法，染色体基因组DNA经HaeⅢ和HinfⅠ两种限制性内切酶酶切后，先进行初级PCR（C1和TELO02）扩增，得到的产物为模板再经过次级PCR（C2和TELO2）扩增，得到的产物用试剂盒回收，并连接到PMD-18-T载体上，转化到JM109受体菌中，从阳性克隆中提取质粒DNA进行PCR和酶切鉴定，确认后进行序列测定，获得了一段243sbp的鸡端粒相关序列。此序列与人第7条染色体基因组末端序列的同源性为63.5%；与鼠端粒旁序列同源性为63.2%；而与MDV基因组序列的BamHⅠ酶切片段76.6%的同源。     

猪胸膜肺炎放线杆菌外膜脂蛋白基因的克隆及序列分析

以国内主要流行的猪胸膜肺炎放线杆菌(APP)QH-1、HN-7菌株的基因组DNA为模板，通过PCR方法扩增出外膜脂蛋白(OML)基因片段，然后将其克隆至pMD18-T载体中，经酶切和PCR鉴定，对阳性克隆进行序列测定。将测序结果分别与标准菌株进行比较，QH—1株的核苷酸序列与血清1、9、11、12型参考株的同源性达99．1％～99．9％；HN-7株的核苷酸序列与血清7、3、4、6型参考株的同源性达97．3％～100％，与其他血清型参考株的同源性较低。     

牦牛外周血单个核细胞抑制消减cDNA文库的构建及部分差异表达分子的克隆分析

为筛选牦牛外周血单核细胞(PBMC)差异性基因,以刀豆素A(ConA)和脂多糖(LPS)联合刺激的PBMC cDNA为实验组,未经诱导刺激的PBMC cDNA为驱动组,利用抑制性消减杂交技术(SSH)构建了丝裂原诱导刺激PBMC的消减文库并对其部分阳性克隆进行了EST序列分析.从消减文库中随机挑取16个阳性克隆,进行PCR鉴定,显示克隆的重组率大于93%,插入片段大小大部分集中在200 bp～1 000 bp之间.随机挑取100个克隆进行测序及同源性分析,初步获得27条差异表达基因片段,其中24个为已知基因,3个为新ESTs序列;随机选择非重复的6个差异表达的序列设计引物,以半定量PCR方法验证其消减效率.结果显示,均从构建的消减文库中扩增到目的片段,其中5个为诱导性差异表达分子,1个为诱导特异性表达分子,说明该文库有较高的质量.本研究应用抑制消减杂交技术构建了牦牛PBMC的差异表达cDNA文库,并高通量克隆鉴定了相关功能基因片段,表明该技术手段有助于快速发现牦牛新功能基因.     

西农萨能羊泌乳高峰期和初期乳腺组织差异表达基因研究

利用抑制性削减杂交和实时定量PCR研究西农萨能羊泌乳高峰期差异表达基因,结果表明成功构建泌乳高峰期和泌乳初期乳腺组织差异表达削减eDNA文库,以GAPDH为指标检测文库削减效率为2^5倍,共获得78个阳性克隆,PCR检测插入片段主要分布在150～1000bp,挑选插入片段不等的30个克隆测序,获得25个有效序列,代表18个基因。对文库中所包含的血清淀粉样蛋白A3（SAA3）,ATP结合盒亚家族G成员2（ABCG2）,心脏型脂肪酸结合蛋白（H-FABP）和黄嘌呤脱氢酶（XDH）进行实时定量PCR检测,发现上述4个基因在泌乳高峰期乳腺组织中的表达水平分别是泌乳初期的17．0,7．7,16．3和1．7倍。结论：构建的消减文库可用于筛选泌乳高峰期差异基因,已鉴定的4个基因很可能是调控产奶量和乳成分变化的候选基因。     

鹅基因组微卫星富集文库的构建与分析

为分离鹅(Anser cygnoides)微卫星序列片段,提取鹅基因组DNA,用Hae III和Rsa I内切酶消化并连接接头,再用接头特异引物进行PCR扩增。扩增产物与生物素标记的(AC)12探针杂交,杂交复合物用链霉亲和素包裹磁珠进行结合,得到单链DNA目标片段。再经PCR扩增,连接pMD19-T载体,转化入感受态大肠杆菌,得到微卫星富集小插入片段DNA文库。用Colony-PCR法筛选获得318个阳性克隆,并进行测序分析。结果表明,所测的318个序列有242个含微卫星序列,197个为有效微卫星序列,其中完全型(perfect)占60.9%,非完全型(imperfect)20.8%,混合型(compound)18.2%。(CA)n重复最为常见。文章为鹅种资源遗传多样性、分子进化、遗传图谱的构建及重要经济性状基因座定位等研究奠定了基础。     

柔嫩艾美耳球虫(E.tenella)杂交株F2 SO7基因ORF的克隆与表达

用反转录聚合酶链式反应(RT-PCR)技术,从柔嫩艾美耳球虫(E.tenella)杂交株F2[F1(JL×HB)×SD]的总RNA中扩增了651 bp的SO7基因ORF。通过T-A克隆技术,将PCR产物克隆至pMD18-T中,转化至受体菌DH5α中,经PCR及酶切鉴定确定阳性克隆pMD18-T-SO7,然后进行序列测定及分析确定成功克隆SO7基因ORF。经DNAStar软件分析核苷酸和氨基酸序列,结果显示扩增出了完整的SO7开放阅读框架,含651个核苷酸,富含AGC重复序列,编码216个氨基酸。与国外株SO7比较,核苷酸序列的同源性为97.7%,有15个核苷酸发生突变,其中9个为有义突变,氨基酸序列的同源性为94.9%。将获得的重组阳性克隆质粒pMD18-T-SO7与融合型表达载体pGEX-6p-1均以BamH I和EcoR I双酶切后。进行连接转化与克隆鉴定。将阳性克隆pGEX-6p-SO7转化至感受态E.coli BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约43.8 Ku融合蛋白带。Western blot分析发现,该蛋白具有E-tenella抗原性,从而为进一步研制E.tenella杂交株F2基因工程疫苗奠定了基础。     

高致病性猪繁殖与呼吸综合征病毒感染猪肺泡巨噬细胞的差异转录基因文库构建

为鉴定高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)HuN4株感染猪肺泡巨噬细胞(PAM)后,细胞内转录发生差异的基因,本研究采用抑制性消减杂交技术,以HP-PRRSV HuN4株感染后48 h的PAM细胞mRNA为实验方(Tester),以未感染的PAM细胞为驱动方(Driver)进行消减杂交,得到了HP-PRRSV HuN4株感染后PAM内转录后发生上调的基因.反之,实验方与驱动方互换进行消减杂交,得到了HP-PRRSV HuN4株感染后PAM内转录后发生下调的基因.将2种杂交所得的差异转录基因经PCR扩增后分别克隆到T载体中并转化大肠杆菌感受态细胞,从而构建了正向(上调)和反向(下调)的差异cDNA文库.随机挑取文库中的多个克隆用PCR方法进行鉴定,结果表明差异文库中的cDNA具有较好的多样性.本研究为下一步的文库克隆的序列测定与差异基因的功能分析奠定了基础.     

大熊猫蛔虫ITS及5.8SrDNA序列的克隆与分析

基于核糖体DNA第一与第二转录间隔序列以及5.8S序列,以分离自广州动物园大熊猫体内的蛔虫为研究对象,用保守引物NC_5和NC_2对核糖体DNA（rDNA）的内转录间隔区ITS-1,ITS-2及5.8S序列进行PCR扩增,扩增后的片段纯化后克隆至pGEM-Teasy载体,重组质粒通过菌液PCR鉴定后,对阳性菌落进行序列测定及分析,鉴定大熊猫蛔虫的种类。结果显示,目的片段总长为910 bp,2个不同样品之间的ITS及5.8S序列没有差异,与GenBank~（TM）中的拜林蛔线虫（Baylisascaris transfuga）、猪蛔虫（Ascaris suum）和人蛔虫（Ascaris lumbricoides）的ITS序列相似性分别为96.6%、82.9%和82.7%。结果表明,此次分离的大熊猫蛔线虫可能为拜林蛔线虫。     

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.     

中华人民共和国农业部公告 第267号

为贯彻落实《兽药生产质量管理规范》(简称《兽药GMP》),进一步推动兽药GMP实施进程,我部制定了《兽药生产质量管理规范检查验收办法》,现予公告。本公告自2003年6月1日起施行。附件:兽药生产质量管理规范检查验收办法二○○三年四月十日第一章 总则 第一条 为推动《兽药生产质量管理规范》(以下简称兽药GMP)的实施,规范兽药GMP检查验收工作,制定本办法。 第二条 农业部负责全国兽药GMP管理和检查验收工作;负责制修订兽药GMP检查验收管理规定;负责兽药GMP检查员队伍建设和监督管理工作,负责国际兽药贸易中GMP互认工作。 …