鸡传染性贫血病毒VP2蛋白单克隆抗体的研制及其识别抗原表位的鉴定

拟制备针对鸡传染性贫血病毒(CIAV) VP2蛋白的单克隆抗体(mAb),为CIAV的诊断和病毒生物学特性研究提供有用制剂.以PCR技术扩增CIAV VP2基因并克隆到原核表达载体pET-32a中,经IPTG诱导表达.以原核表达的融合蛋白免疫BALB/c小鼠,利用杂交瘤技术研制并筛选分泌抗CIAV VP2蛋白mAb的阳...     

牛疱疹病毒Ⅰ型gB基因的原核表达及产物的抗原性分析

采用PCR方法，以牛疱疹病毒Ⅰ型基因组DNA为模板扩增gB基因，克隆至pGEM—T载体。将以克隆质粒pGEM-T-gB为模板扩增的gBⅠ、gBⅡ和gBⅢ基因片段分别连接至原核表达载体pET32a。获得的重组质粒分别转化感受态细胞BL21（DE3），在IPTG诱导下表达。经SDS-PAGE鉴定，gBⅠ、gBⅡ和gBⅢ基因片段在大肠埃希氏菌BL21（DE3）中以融合蛋白形式获得了表达。以磁化组氨酸蛋白纯化系统对融合蛋白纯化后，Western-blotting和ELISA分析结果表明，这些融合蛋白均与BHV-1标准阳性血清反应，可作为抗原用于牛传染性鼻气管炎ELISA检测方法的建立。     

鸡传染性贫血病毒vp1基因的克隆表达及抗原性分析

从组织病料中提取CAV核酸,根据GenBank上发表的序列设计两对引物,分别扩增出vp1的139 bp～83 bp和547 bp～1337 bp两个基因片段,然后分别将其充隆到原核表达载体PMXB10上,经PCR和酶切鉴定,证明成功构建了重组表达载体PMXB10-vp1C和PMXB10-vp1D。在0.3 mM的IPTG诱导下,融合蛋白在大肠杆菌ER（2566）以分泌型得到大量表达,经大量表达后,用几丁质柱挂柱切割纯化后,得到切割蛋白E、F,在Western blot免疫分析印迹中,两组融合蛋白和切割蛋白与CAV阳性血清均能发生特异性反应。用ELISA方法检验,纯化的两组蛋白与CAV阳性血清均能发生特异性反应。用两组蛋白免疫SPF鸡后,用全病毒ELISA试剂盒检测血清呈阳性,表明两组蛋白均可诱发机体产生抗CAV的抗体。     

犬细小病毒VP2基因的原核表达及间接ELISA方法的建立

根据GenBank上发表的犬细小病毒（CPV）VP2蛋白基因序列，设计并合成1对引物，通过PCR扩增VP2基因全长。将其克隆到pET一30a载体中，构建了原核表达载体pET—VP2，转化大肠杆菌Rosetta，表达并纯化了重组蛋白，Western—blotting证明该重组蛋白具有免疫原性。利用重组蛋白为抗原，通过方阵试验确定了包被抗原的最佳包被量为15．8ng／孔，血清的最佳稀释倍数为1：100，建立了检测CPV血清抗体的间接ELISA方法。     

猪细小病毒VP2的原核表达与多克隆抗体制备

克隆猪细小病毒(PPV)VP2基因全长1 740bp,构建其原核表达载体后进行蛋白的表达与纯化,接种家兔制备多克隆抗体。用PCR方法扩增PPV VP2全长基因,扩增产物克隆至表达载体pET-32a并转化至E.coli BL21(DE3)感受态中。分别用不同诱导时间、IPTG浓度、温度诱导表达VP2重组蛋白,经SDS-PAGE电泳切胶回收纯化目的蛋白。对家兔进行3次免疫后,采集血清制备抗体。PCR扩增得到1 740bp的VP2基因片段;构建原核表达载体pET-32a-VP2,经37℃,IPTG浓度1.0mmol/L诱导表达4h可得分子质量大小约为82ku目的蛋白;间接ELISA检测抗体效价可达1∶12 800;Western blot证明所制备的抗体能够有效的应用于PPV VP2抗原的检测。本研究成功构建了表达PPV VP2基因的原核载体,获得了VP2蛋白,制备了兔抗多克隆抗体,为建立检测PPV VP2蛋白ELISA方法奠定了基础。     

鸡贫血病毒vp2基因的表达、纯化及重组蛋白的鉴定

根据GenBank上已发表的鸡贫血病毒Cux-1株基因组序列设计并合成了1对特异性引物,通过PCR扩增出vp2基因,将扩增出的片段克隆到pGM-T载体上,通过测序分析证实该片段与鸡贫血病毒Cux-1株的vp2基因序列一致。将克隆的W2基因亚克隆到原核表达载体pET-32a（＋）上并转化至大肠杆菌BL21（DE3）中,用IPTG诱导表达,表达的融合蛋白进行SDS-PAGE和Westernblot分析,并用镍柱在天然状态下进行了纯化。结果表明,表达的融合蛋白分子量约为45ku,能与鸡贫血病毒阳性血清发生反应。     

鸡传染性贫血病毒VP2基因的原核表达及兔抗血清的制备

为了用大肠杆菌原核表达系统表达鸡传染性贫血病毒(CIAV)VP2基因片段,并制备其兔抗血清,试验以CIAV M9905株基因组DNA为模板,通过PCR扩增VP2基因,与p ET28a(+)载体连接后转化DH5α获得阳性重组子,将其转化E.coli BL21(DE3)菌,IPTG诱导表达出His-VP2重组蛋白,经Ni-NTA树脂纯化后免疫新西兰白兔制备兔抗血清,采用IFA和Western-blot检测该血清的特异性,ELISA测定其效价。结果表明:重组His-VP2融合蛋白的分子质量约为36 ku,以包涵体形式存在。制备的兔抗VP2血清效价在1∶6 400以上,可特异性结合真核表达的VP2蛋白,表明其反应原性较好。说明试验成功表达VP2蛋白,并制备出兔抗血清,可用于该蛋白与病毒其他蛋白之间的相互作用的进一步研究。     

金黄色葡萄球菌纤连蛋白结合蛋白A基因的克隆及其原核表达

根据GenBank中纤连蛋白结合蛋白A基因（fnbA）序列设计了1对特异性引物，以金黄色葡萄球茵基因组DNA为模板，进行PCR扩增；结果获得了3600bp的DNA片段。将PCR产物克隆至pGEM T easy载体中，成功地构建了克隆质粒pGEM—fnbA。以HindⅢ和XhoⅠ双酶切pGEM-fnbA和pET28a（＋），将纯化的基因fnbA亚克隆至pET28a（＋）中，构建了原核表达质粒pET28a-fnbA，并将其转化至E．coli BL21（DE3）感受态细胞中，经1mmol／LIPTG诱导和SDSPAGE分析，在约165ku处出现了与预期目的蛋白一致的外源蛋白带。Western—blotting分析表明，该蛋白具有金黄色葡萄球菌的抗原性。     

鸡传染性贫血病毒衣壳蛋白的原核表达及免疫原性分析

将从组织病料中提取的鸡传染性贫血病毒(chicken infectious anemia virus,CIAV)核酸进行PCR扩增,获得vp1基因,将其克隆到表达载体pET32a(+)中,构建了CIAV vp1基因重组质粒,命名为pET32a-vp1。将 pET32a-vp1转化E.coli plys,重组菌经IPTG诱导,SDS-PAGE分析结果表明,vp1基因在大肠杆菌中成功表达。纯化的蛋白质作为包被抗原,ELISA鉴定结果表明具有良好的抗原性。本试验结果为进一步研究用重组抗原制备CIAV ELISA诊断试剂盒奠定了基础。     

鸡贫血病病毒VP1基因的克隆分析

目的:探讨鸡贫血病病毒VP1基因的克隆方法与效果。方法:从组织病料中提取鸡贫血病病毒核酸,根据GenBank上发表的序列设计两对引物,扩增基因片段,然后分别将其克隆到原核表达栽体pMXB10上,经PCR和酶切鉴定。结果:PCR产物经琼脂糖凝胶电泳分析,分别在690BP和790bp有特异性产物出现。对重组质粒PMXB10VP1A和PMXB10VP1B进行酶切鉴定,与预期结果相同。结论:鸡贫血病病毒VP1基因的克隆成功有效。     

Anaesthesia and analgesia of the donkey and the mule

The number of donkeys and mules throughout the world is stable, and awareness of their use and concern for welfare, pain recognition and treatment are receiving increasing veterinary interest. Therefore, accurate information about anaesthesia and analgesia in donkeys and mules is important to ever more equine practitioners. Since donkeys are physiologically and pharmacologically different from horses, knowledge on species specific aspects of anaesthesia and analgesia are very important. Mules combine elements from both donkey and horse backgrounds, leading to great diversity in size, temperament and body type. Physiologically, they seem to resemble horses more than donkeys. This review highlights the current knowledge on various anaesthetic and analgesic approaches in donkeys and mules. There is still much information that is not available about donkeys; in many circumstances, the clinician must use available equine information to treat the patient, while monitoring carefully to observe for differences in response to therapy compared to the horse.     

Ultrasonographic examination of the caecum and the proximal and spiral ansa of the colon of cattle

The large intestine of 10 cows was examined from the right abdominal wall with a 3.5 MHz linear transducer. The cows were then slaughtered, and the organs were re-examined in a water bath. The caecum was visualised from the middle region of the abdominal wall. It ran caudo-cranially, varied in diameter from 5.2 to 18.0 cm and was situated immediately adjacent to the abdominal wall. The lateral wall of the caecum appeared as a thick, echogenic, crescent-shaped line. It could be visualised as far cranially as the 12th intercostal space. Although its junction could not be identified, the proximal ansa of the colon was recognised on the basis of its anatomical position and its diameter, which was smaller than that of the caecum. The spiral ansa of the colon and the descending colon were situated dorsal to the caecum and could be identified by moving the transducer horizontally along the abdominal wall to the last rib. The spiral ansa of the colon was situated ventral to the descending colon, and its walls appeared as thick echogenic lines. In a contracted state, the spiral colon had the appearance of a garland.     

Effects of age and diet on the development of the pancreas and the synthesis and secretion of pancreatic enzymes in the young pig

Effects of age and diet composition on amylase, trypsin and chymotrypsin activities in the pancreas and intestinal contents, pancreas weights and body weights were determined from birth to 56 d. A total of 120 pigs, five to seven pigs/litter from 18 litters, were slaughtered at birth, 14, 27, 29, 31, 42 and 56 d. Litters were allotted to dietary treatments (corn-soy, A; corn-soy + 20% dried whey, B; corn-soy + 5% lard, C) and offered these diets as creep feed at 14 d. All pigs were weaned at 28 d, placed in elevated nursery pens and fed their respective diets. Total activities of amylase, trypsin and chymotrypsin in the pancreas and small intestine increased (P less than .05) with age. Both trypsin and amylase activities, measured per kilogram body weight or gram pancreas weight, were low at 29 d in the intestine and increased to 56 d. Pigs on diet B had the highest level of trypsin and chymotrypsin in the intestinal contents (P less than .05). Trypsin activity in the pancreas (units/kg body weight) was lowest (P less than .05) for pigs on diet B and highest (P less than .05) for those on diet C (units/g pancreas and units/kg body weight). Amylase activity (units/kg body weight) was lower (P less than .05) in the pancreas for pigs on diet B than for those on diets A and C. Pigs on diet A had lower (P less than .01) intestinal amylase activities than those on diets B and C. Enzyme activities in the intestinal contents and pancreas were low following weaning. In the pancreas, activities decreased at 31 d.(ABSTRACT TRUNCATED AT 250 WORDS)     

加强兽药研究开发提升行业科技创新水平

提升行业科技创新水平为主题,回顾和分析了我国新兽药研究开发的历程和研究现状及面临的形势,并围绕创新与发展的要求,提出了加强兽药研究开发的对策及建议。     

The behavior and the husbandry conditions of small laboratory animals--possibilities and limits of the evaluation of the appropriateness and fairness of the treatment of animals

Ethological studies are important means to evaluate the behaviour of small laboratory animals (mouse, rat, guinea-pig and rabbit) and their housing conditions. At any rate, the only sparse range of body expressions, the smallness and preference of night activity of these animals and the erratic occurrence of essential behaviour patterns make the investigations more difficult. On the other hand, the standardization of laboratory housing and the genetic definition of these animals turn out to be favourable. By this way, behaviour-environment-relationships are suitable for evaluation. Taking into consideration methodical and biological restrictions, the specific activities such as fight, relaxed resting behaviour and play as well as unbalanced behaviour patterns and stereotypes can be taken as means of evaluation. Particularly in mice and rabbits, upright position and space consuming activities will be possibly restricted by cage sizes.     

Relation between the concentration of sodium and potassium in the erythrocytes of dairy cows and the concentration of these elements in the plasma and urine and the level of blood glucose

Blood and urine samples of 180 dairy cows from 12 herds were examined. Sodium and potassium concentrations were determined in erythrocytes, whole blood, plasma, urine, and glucose concentration in blood: the interrelations were compared. Sodium and potassium concentrations in erythrocytes were 85.15 +/- 11.45 mmol/l, and 25.93 +/- 7.81 mmol/l, respectively. A statistically significant relation was found between sodium and potassium concentrations in erythrocytes (r = 0.3467+++) and the content of electrolyte in blood cells and in whole blood (Na: r = 0.5336+++; K: r = 0.3561+++). No statistically significant relation of intraerythrocyte concentration of both electrolytes was confirmed with respect to the other characteristics (sodium and potassium concentrations in plasma and urine, and glucose concentration in blood). In the conditions of routine laboratory diagnostics in clinically healthy cows, determinations of intraerythrocyte sodium and potassium do not broaden possibilities of evaluating the metabolic state of these electrolytes and cannot be used as the characteristics of energy insufficiency. The cannot replace the analyses performed in serum and urine.     

貉,蓝狐,银狐的胃及十二指肠内嗜银细胞形态分布的研究

本文应用Ｆｏｒｔａｎａ—Ｍａｓｓｏｎ嗜银颗粒染色法研究了貉、蓝狐、银狐的胃及十二指肠内嗜银细胞的形态分布。结果表明：三种动物胃幽门腺中，嗜根细胞数量最多，呈锥状，嗜银颗粒较粗，密度大、染成深黑色。在胃底及胃贲门腺区、嗜银细胞数量少。在十二指肠部嗜银细胞呈蝌蚪形，嗜银颗粒细，呈黑褐色，密度较小。嗜银细胞的分布密度在三种动物中具有一定的差别。     

Liquid flow and capacity of the caecum and colon of the horse

The rate of flow of fluid from the caecum and from the large colon was measured in four Shetland-type ponies fed a hay diet. In two ponies with cannulas in the caecum and at the origin of the right ventral colon, a continuous intracaecal infusion of a solution of chromium EDTA was used and samples were obtained from the cannula at the origin of the right ventral colon. Based on four determinations, the liquid flow from the caecum was 54.2 +/- 1.89 litres d-1. In the other two ponies with cannulas in the origin of the right ventral colon and near the end of the right dorsal colon, a continuous infusion of chromium EDTA was made into the right ventral colon and samples were obtained from the right dorsal colon. The flow rate towards the end of the dorsal colon was 49.4 +/- 1.25 litres d-1, based on four determinations. The capacities of the caecum and large colon after death were 7.0 +/- 0.8 and 17.7 +/- 3.7 litres, respectively.     

Conduction velocities and reflexes of the proximal and distal parts of the saphenous nerve of the dog

The maximal conduction velocities of compound-action potentials in the proximal and distal parts of the saphenous nerve were determined by averaging potentials evoked and recorded through needle electrodes. Antidromic, triphasic compound-action potentials unipolarly recorded from the distal part of the saphenous nerve were of the same minimal latency as potentials having 4 phases which were recorded bipolarly from the same site. However, the unipolarly recorded potentials were of greater amplitude. Monophasic compound-action potentials were recorded through bipolar chlorided silver electrodes from the surface of fascicles of the distal part of the saphenous nerve. The maximal conduction velocity of these potentials was in agreement with the conduction velocity of compound-action potentials of the distal part of the saphenous nerve which were evoked and recorded through subcutaneous needle electrodes. The specificities of the stimulating and recording sites were verified by recording before and after the saphenous nerve was cut between the stimulating and recording sites. Mean conduction velocities were 62.3 +/- 2.0 m/s for the distal part of the saphenous nerve and 66.3 +/- 2.2 m/s for the proximal part of the saphenous nerve. Reflex-evoked muscle activity was elicited in the ipsilateral tensor muscle of the fascia lata and semimembranous muscle after electrical stimulation of the saphenous nerve through subcutaneous needle electrodes. The effects of various stimulus intensities on the latency and duration of these reflex-evoked muscle potentials were determined.