口蹄疫病毒VP1 T、B细胞表位与大肠杆菌肠毒素融合蛋白的免疫应答

本实验比较了O型口蹄疫病毒（FMDV）VP1双拷贝T细胞表4i（aa21～aa40）、B细胞表位（aa141～aa160）与肠毒素大肠杆菌肠毒素融合后蛋白2020-B-2020和2020-B-2020-STI的免疫原性。动物实验表明，2种融合蛋白具有良好的免疫原性，其中2020-B-2020-STI免疫血清抗体水平优于2020-B-2020；免疫豚鼠在低浓度FMDV刺激下能够产生特异性T淋巴细胞增殖反应，说明2种融合蛋白能诱导机体产生FMDV特异性细胞及体液免疫反应。2020-B-2020和2020-B-2020-STI融合蛋白免疫雌鼠能够抵抗大肠杆菌强毒株攻击，免疫保护率分别为：2020-B-202060％／1．5MLD，2020-B-2020-ST／100％／1．5MLD；2020-B-2020-STI免疫血清中具有STI中和抗体。且融合蛋白不具STI毒性。ELISA实验结果显示，2020-B-2020和2020-B-2020-STI能与霍乱毒素CTB抗体特异性结合。实验结果表明，2020-B-2020-STI具有开发成为口蹄疫及肠毒素大肠杆菌疫苗的应用价值。     

亚洲Ⅰ型口蹄疫病毒双拷贝VP1基因的串联表达及其免疫原性

口蹄疫病毒（FMDV）VP1基因含有T细胞和B细胞表位,是口蹄疫病毒的主要免疫原性基因。作者将亚洲Ⅰ型FMDV VP1基因首尾串联构建双拷贝的VP1基因（2VP1）,实现双拷贝VP1基因的原核表达,表达的重组蛋白（GST2VP1）经Sephadex-G200分子筛层析纯化后,Western blot证实其有反应原性,动物试验表明重组蛋白在加强免疫后能够产生和灭活疫苗相当的ELISA抗体和中和抗体;本试验结果为亚洲Ⅰ型FMDV免疫原性研究及GST2VP1蛋白的进一步应用奠定了基础。     

O型口蹄疫病毒VP1 T细胞与B细胞表位基因双拷贝串联表达产物的免疫应答

以一株O型口蹄疫病毒(FMDV)外壳蛋白VP1基因为模板,合成与细胞免疫及体液免疫相关抗原表位肽基因:21-40肽(20AA)和141-160肽(20AA)基因序列,运用基因工程技术构建了含有串联结构21-40(20AA)～141-160(20AA)～21-40(20AA)～141-160(20AA)的2020-2020VP1融合基因表达载体r2020-2020,转化宿主菌BL21(DE3)RIL后诱导表达,表达产物经SDS-PAGE及Western Blot分析显示重组融合蛋白的分子量约为18Ku.动物实验表明,较小剂量的融合蛋白就能诱导豚鼠产生特异性T淋巴细胞增殖反应及抗FMDV中和抗体,证明该融合蛋白可同时激活细胞免疫及体液免疫反应,具有开发成为抗FMDV疫苗的应用价值.     

重组表达口蹄疫病毒T细胞表位猪细小病毒病毒样颗粒的制备

为增强表位疫苗的免疫原性,本研究将O型口蹄疫病毒(FMDV)VP1中编码T细胞表位肽(aa 21～aa 40)序列连接于猪细小病毒(PPV)VP2的5'端,并插入到杆状病毒供体质粒pFastBac HTA中,构建成重组转座载体pFastBac-FMDV-VP1-PPV-VP2,转化含有穿梭载体Bacmid的E.coli DH10Bac中,经蓝白菌落筛选获得重组杆状病毒表达质粒rBac-FMDV-VP1-PPV-VP2,转染昆虫草地夜蛾(Sf9)细胞,并在Sf9中进行了表达。电镜观察显示,表达的重组蛋白能够自我组装成病毒样颗粒rPPV∶VLP(FMDV)。用间接免疫荧光试验(IFA)、western blot分析表明,表达产物具有与PPV VP2天然蛋白相似的免疫原性。小鼠免疫实验证实,在低浓度FMDV抗原下能够产生特异性T淋巴细胞增殖反应;而且该病毒样颗粒能诱导机体产生抗FMDV特异性细胞免疫反应。     

Asial型口蹄疫病毒VP2蛋白B细胞线性表位的鉴定

为了对Asial型口蹄疫病毒VP2蛋白进行抗原表位作图,设计了7个相互重叠15个氨基酸、覆盖整个VP2蛋白的重叠短肽,经克隆并在大肠杆菌中实现了融合表达.用Asial型口蹄疫感染血清对7个融合蛋白进行免疫印迹分析,结果初步鉴定出VP2蛋白B细胞线性抗原表位位于氨基末端的第1-44氨基酸区域,并且证实O型、A型、C型口蹄疫标准血清和感染SAT2型口蹄疫康复期的牛血清也能识别融合蛋白F1(1-44 aa).在此基础上,针对F1(1-44 aa)短肽,设计了6个相互重叠5个氨基酸的短肽片段,进一步鉴定出了VP2蛋白B细胞线性表位位于氨基末端的第6-15氨基酸区域.     

口蹄疫病毒非结构蛋白2C基因主要表位区的表达及活性检测

根据测定的口蹄疫病毒（FMDV）2C基因序列,设计了一对特异性的表达引物,用于扩增2C基因5′-端174bp和3′-端279 bp连接片段,该区含有较丰富的B细胞表位编码序列。扩增片段通过NcoⅠ和SalⅠ酶切位点插入表达载体pET-30a。序列测定表明,目的基因片段连接正确,按正确的读码框插入表达载体中。重组表达质粒转化BL21（DE3）pLys,经IPTG诱导表达目的蛋白。表达产物经SDS-PAGE和Western Blotting检测表明,2C基因主要表位区成功地在大肠杆菌表达,表达产物为约23 ku的融合蛋白,能够与FMDV感染动物血清发生特异性反应,而不与健康和灭活疫苗免疫动物血清反应。该研究为建立鉴别FMDV自然感染动物和灭活疫苗免疫动物的酶联免疫转印（EITB）方法提供了所需的材料。     

口蹄疫病毒VP2基因的真核表达及其产物抗原性的检测

利用PCR技术，扩增出口蹄疫病毒（FMDV）VP2基因，并克隆到杆状病毒转移载体pBlueBacHis2A上。用重组质粒pB-VP2与重组病毒同时转染sf9昆虫细胞，获得了重组病毒。经过蚀斑筛选纯化后，感染sf9细胞，表达VP2融合蛋白，分子质量为33ku左右。以牛抗O型FMDV血清为第一抗体，通过Western-blotting和Dot-ELISA鉴定，说明VP2基因在真核表达系统中获得正确表达，且可以与牛抗。型FMDV血清发生特异性反应。     

O型口蹄疫病毒结构蛋白VP1的原核表达与抗原性分析

研究分析了O型口蹄疫病毒(FMDV)结构蛋白VP1与当前猪FMDV疫苗血清的免疫反应性.将VP1基因克隆至原核表达载体pET32c,并在大肠埃希菌BL21中得到了表达,Western blot分析表明该重组蛋白与豚鼠O型FMDV标准阳性血清具有良好免疫反应性.目的蛋白经纯化后用ELISA分析其与猪疫苗血清的免疫反应性,结果显示该重组VP1蛋白(rVP1)只能与部分O型FMDV疫苗血清反应.推测当前使用的不同O型FMDV疫苗毒株在VP1重要中和抗原位点G-H环(134 aa～158 aa)与C末端(200 aa～213 aa)存在较大差异.     

非致病大肠杆菌鞭毛与O型FMDV结构蛋白VP1嵌合体的免疫效力评估

为探讨以非致病性大肠杆菌鞭毛蛋白为载体展示口蹄疫病毒(FMDV) VP1蛋白嵌合体(Fn-VP1-Fc)的免疫效力为目的,筛选2月龄FMDV阴性仔猪,颈部肌肉注射,分别于免疫前、免疫后第14、28和60天采集血清,用液相阻断ELISA (LPB-ELISA)测定IgG滴度。分别于免疫前和免疫后第28天,采集鼻腔拭子和肛拭子,间接ELISA测定IgA滴度。结果显示,疫苗接种第14天,免疫Fn-VP1-Fc疫苗产生了可以检测到的FMDV特异性抗体;第28和60天,FMDV特异性抗体持续升高;第60天,产生了可以检测到的特异性IgA,部分猪只滴度可达1∶160,而对照疫苗未检测到IgA。结果表明,Fn-VP1-Fc嵌合免疫原不仅能促进FMDV全身特异性抗体产生,也在一定程度上促进局部IgA分泌。     

肝片吸虫Fh8标签融合表达口蹄疫O型病毒结构蛋白

口蹄疫病毒(foot and mouth disease virus,FMDV)的结构蛋白VP1难以实现体外的高效表达。本研究为获得良好免疫原性的VP1蛋白,选用肝片吸虫Fh8小肽段作为新型融合表达标签,构建重组Fh8-VP1免疫原,以期获得大肠杆菌中的高效表达。分别合成VP1的20~40aa和129~169aa之间的核苷酸片段,并用2个甘氨酸(GG)连接2个片段,合成的核苷酸片段(ΔVP1)克隆入Fh8标签之后,构建重组菌BL21(DE3)/pColdⅠ-Fh8-ΔVP1,SDSPAGE分析蛋白表达形式,Western blot方法检测重组His-Fh8-ΔVP1的抗原性。结果显示,成功构建重组质粒BL21(DE3)/pColdⅠ-Fh8-ΔVP1,并在大肠杆菌BL21(DE3)中获得高效表达,且以包涵体形式存在于菌体蛋白,可与猪源FMDV阳性血清发生特异性反应。     

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.