伪狂犬病病毒Ma株基因组3.4kb及4.4kbBamHI片段的克隆与鉴定

本试验采用BamHI酶切伪狂犬病病毒 (PRV)Ma株基因组DNA ,电泳回收 3 4kb和4 4kb片段 ,快速克隆了伪狂犬病病毒Ma株BamHI 3 4kb和 4 4kb片段到质粒 pUC1 9中 ,对其进行部分序列测定并与Genebank序列进行同源性分析 ,结果表明BamHI 3 4kb片段包含UL50等基因 ,与Ka株UL50基因同源性为 87% ,BamHI 4 4kb片段包含RSP40及PK等基因片段 ,与Ka株RSP40基因同源性为 98%。Ka株BamHI 3 4kb片段包含TK基因 ,而包含UL50基因的BamHI片段为 7 4kb。结果表明 ,PRVMa株BamHI酶切位点发生了漂移     

伪狂犬病病毒Ea株gM和gN基因的克隆与结构分析

gM和Ng是最近经经典免疫沉淀法确定的伪狂犬病病毒（PRV）第三对异源糖蛋白二聚体。根据PRV国外Ka株的核苷酸序列，设计两对分别包含gM和Ng完整编码的特异性引物，以PRV国内地方分离株（Ea株）的细胞感染物为模板，PCR扩增出大小约1．2kb和0．3kb的特异性片段。将扩增物克隆到杆状病毒转座载体pEastBacl中，酶切鉴定证实后进行序列测定。结果表明：Ea株gM、Ng基因分别编码394和98个氨基酸，与Ka株的同源性分别为98．4％和97．6％。DNATool和DNASIS软件对gM、Ng进行二级结构预测，发现gM存在8个潜在跨膜区和一个N－糖基化位点，是典型的能反复多次跨膜的Ⅲ型糖蛋白；gN具有N－端信号肽序列、C－端跨膜区和潜在0－糖基化位点，属Ⅰ型糖蛋白。上述结果为下一步深入研究gM、Ng的相互作用位点及二聚体的功能奠定了功能。     

伪狂犬病病毒闽A株gE基因去信号肽片段的克隆与序列测定

根据已经发表的伪狂犬病病毒（PRV）Rice株的gE基因序列,设计并合成了1对引物,通过PCR方法扩增到了PRV闽A株糖蛋白gE基因除信号肽以外的全部编码区段,并克隆到pMD18-T载体中,转化大肠杆菌XL1-blue菌株,重组质粒pMD18-T-FL经酶切和PCR鉴定证实后,进行了序列测定。结果表明,重组质粒pMD18-T-FL含有PRV闽A株糖蛋白gE基因除信号除信号肽以外的全部编码区段,长1674bp。序列比较分析表明,此区段与PRV Rice株相应区段的核苷酸序列同源性为97．5％,氨基酸序列同源性为94．8％。     

伪狂犬病病毒Bartha-K61株UL49基因的克隆和序列分析

根据已经发表的伪狂犬病病毒(PRV)国内Ea株UL49基因序列,设计并合成了1对引物,通过PCR方法扩增到了PRV Bartha-K61株UL49基因的编码区,并克隆到pMD18-T载体中.重组质粒pMD-UL49经XhoI酶切和PCR鉴定后,进行了序列测定和分析.结果表明,重组质粒pMD-UL49含有PRV Bartha-K61株UL49基因的编码区.同源性分析显示,PRV Bartha-K61株UL49基因序列与国外Kaplan株、国内Ea株相应基因的核苷酸同源性分别为98.9 %和94.1 %,推导的氨基酸序列同源性分别为96.7 %和87.2 %.     

伪狂犬病病毒上海株EP0基因的序列分析及其表达载体的构建

以伪狂犬病病毒上海株 (PRV SH)细胞感染物为模板 ,PCR扩增出 1 2 3kb的EP0基因完整编码区片段 ,将该基因片段克隆到pGEM T easy中 ,经过双脱氧末端终止法序列测定 ,并同国内的PRV Ea株进行同源比较 ,发现PRV SH株EP0基因存在多处突变和一处插入。进一步将该片段插入到原核表达载体 pET32a (+)的His Tag下游 ,构建了的原核表达质粒 pETEP0 ,为今后深入研究该基因的表达及其功能奠定了基础。     

伪狂犬病病毒Fa株gD基因的克隆及序列分析

本研究从含有伪狂犬病病毒(Pseudorabies virus，PRV)Ea株gD基因BamHI 6.6Kb片段的重组质粒pUCB7中分别亚克隆gD基因的上、下游片段，并对上游片段进一步改造，去掉gD基因上游的非编码序列，构建了含完整gD基因编码区的重组质粒pBRgDI，并利用基因内部的限制性内切酶位点，用内切酶KpnI和SalI酶切分析，证实了gD基因的可靠性和完整性。同时构建了测序质粒pSKgDSB和pSKgDS，并用双脱氧终止法进行测序。将测序结果与国外的Rice毒株进行比较，发现Ea株gD基因核苷酸序列有多处点突变和一处插入突变，在编码氨基酸残基水平上也有差异。     

PRV LA株gD基因的序列测定及其重复高变区的发现

对猪伪狂犬病病毒鲁A株(PRVLA株)gD基因进行了克隆和序列测定，结果表明：在测序的l453bp的DNA序列中包括着1个l203bp的ORF(即gD基因)，它编码400个氨基酸组成的多肽；在整个gD基因的ORF内PRVLA株与PRV Ea株、Hulbei株、Rice株、NIA-3株、Kaplan株的gD基因比较，核苷酸的同源性分别为98．3％、98．3％、98．0％、98．1％、98．6％，氨基酸的同源性分别为97．8％、97．8％、97．5％、98．1％、98．6％；发现PRVLA株gD基因与Ea株、Hubei株、Rice株、NIA-3株、Kaplan株的gD基因均在802～837nt处有1个C(A)GGCCC的重复高变区，其对应的是gD267～279位氨基酸残基Arg—Pro的重复高变区。正是该重复高变区的碱基缺失或插入使得PRVgD的ORF在l194～l215nt间变化，gD前体的氨基酸残基为398～404个。     

伪狂犬病病毒Ea株gD基因的克隆及序列分析

本研究从含有伪狂犬病毒（Pseudorabies virus,PRV)Ea株gD基因BamHI 6.6Kb片段的重组质粒pUCB7中分别亚克隆gD基因的上，下游片段，并对上游片段进一步改造，去掉gD基因上游的非编码序列，构建了含完整gD基因编码区的重组质粒，pBRgDI，并利用基因内部的限制性内切酶位点，用内切酶KpnI和SalI酶切分析，证实了 gD基因的可靠性和完整性，同时构建了测序质粒pSKgDSB,pSKgDS，并用双脱氧终止法进行测序，将测序结果与国外的Rice毒株进行比较，发现Ea株gD基因核苷酸序列有多处点突变和一处插入突变，在编码氨基酸残苦水平上也有差异。     

伪狂犬病病毒Ea株Us9基因的克隆与序列分析

对含伪狂犬病病毒Ea株BamH17片段的质粒pUC6.6进行亚克隆，构建了仅含Us9基因约0.6kb片段的重组质粒pSKMN0.6。双脱氧末端终止法进行双链测序，结果表明：Us9存在2种可能的同C-末端的编码形式，分别编码106或98个氨基酸残基，Us9基因与其下游的Us2(28K)基因之间存在约200bp的非转录区，将Ea株Us9基因序列同国外Rice株进行没源比较，发现二者在组成和结构上存在多处保守序列，尤其是潜在的酪氨酸磷酸化位点，C-端疏水性氨基酸以及由连续6个带正电荷的精氨酸残基组成的核定位信号序列，但在N-糖基化位点以及丝氨酸含量上存在较大差异。     

PK and PK/PD of doxycycline in drinking water after therapeutic use in pigs

A commercial doxycycline formulation was administered in drinking water to 12 pigs at the recommended dose of 10 mg/kg daily for 5 days. The mean plasma concentration at steady-state was 1.37 +/- 1.21 microg/mL, which was reached at 68 +/- 27.2 h postadministration. Absorption and elimination half-life values were 7.20 +/- 2.42 and 7.01 +/- 2.10 h, respectively. Most plasma concentrations during dosing were higher than the minimum inhibitory concentrations (MICs) described for the main porcine bacterial pathogens of the respiratory tract (Pasteurella multocida, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica and Mycoplasma hyopneumoniae). It is concluded that when pigs were treated with doxycycline in drinking water at the recommended rate, therapeutically effective concentrations were achieved throughout the treatment period, supporting the clinical use of this tetracycline in the control of respiratory infections. However, inter-animal differences were marked.     

弓形体RH株在3种传代细胞中增殖情况的比较

为建立有效适用的弓形体体外培养方法,本试验利用Vero、PK15、RK13 3种传代细胞和DMEM、MEM、RPM I1640三种培养液分别对RH株弓形体速殖子进行培养,并比较了虫体的增殖和活力情况。结果显示,利用Vero细胞DMEM培养液培养弓形体速殖子,虫体增殖快、活力强,是理想的培养方法。     

PK结肠溶胶囊对犬结肠炎的疗效评价

为评价PK结肠溶胶囊对犬结肠炎的治疗效果,探索更安全有效的治疗药物,以16只中华田园犬作为试验动物制作急性溃疡性结肠炎模型,随机平均分为4组,试验组分别口服PK结肠溶胶囊1颗/kg(正常剂量组)、2颗/kg(2倍剂量组)及柳氮磺吡啶结肠溶胶囊(阳性药物组,1颗/kg),测定给药后犬血液白细胞总数(WBC)、C-反应蛋白(CRP)参数变化,用内窥镜观察肠黏膜愈合状况。结果:2倍剂量组和阳性药物组给药后第4dWBC恢复到正常范围,正常剂量组给药后第7d未恢复到正常范围;2倍剂量组给药后第7dCRP恢复到正常范围,正常剂量组和阳性药物组未恢复到正常范围;内窥镜观察肠黏膜愈合状况疗效最好的是2倍剂量组。结论:PK结肠溶胶囊以2颗/kg剂量治疗犬结肠炎疗效明显,效果优于1颗/kg的正常剂量和柳氮磺吡啶。     

猪圆环病毒Ⅱ型Rep基因在PK15细胞中的表达及特性

为研究猪圆环病毒Ⅱ型(PCV2)Rep基因在PK15细胞中的表达特性，通过PCR方法克隆了PCV2杭州株(HZ0201)Rep基因全长945bp片段，与真核表达栽体pCI—neo构建为重组质粒pCI-PCV2-Rep。pCI-PCV2-Rep质粒转染PK15细胞后48h，通过RT-PCR可检测到PCV2 Rep mRNA的转录；用猪PCV2多抗血清作间接免疫荧光试验，可检测到Rep基因表达产物。在表达量低的细胞中，PCV2 Rep蛋白主要位于PK15的细胞浆，在表达量高的细胞中，细胞浆和细胞核中均含有大量的Rep蛋白，表明Rep对PK15细胞的细胞浆和细胞核的亲嗜性没有明显差别。     

猪细小病毒在PK15细胞中增殖规律的研究

猪细小病毒毒株在不同细胞系上的生长情况不尽相同,如不能掌握其规律,将难以生产出优质的疫苗。对PPV（长春株）在PK15和ST细胞上生长情况进行了研究,在PK15细胞系中重点比较了不同的病毒接种方法、病毒接种量和病毒收获时间等因素对病毒产量的影响。研究结果显示：本毒株在PK15和ST两种细胞系上的病变特征不同,但生长规模相似。在PK15细胞上按2％接种量、分步接种、80h时收获病毒,最终得到的病毒滴度（TCID50）最高。提示这是一种比较好的生产方案。然而,进一步的大规模培养试验是有必要的。     

Pharmacokinetics and pharmacodynamics of ketoprofen in calves applying PK/PD modelling

The pharmacokinetics (PK) and pharmacodynamics (PD) of ketoprofen (KTP) were studied in calves following intravenous administration of the drug racemate at a dose rate of 3 mg/kg. To evaluate the anti-inflammatory properties of KTP, a model of acute inflammation, consisting of surgically implanted subcutaneous tissue cages stimulated by intracaveal injection of carrageenan, was used. No differences were observed between disposition curves of KTP enantiomers in plasma, exudate or transudate. This indicates that in calves KTP pharmacokinetics is not enantioselective. S(+)- and R(-)- KTP each had a short elimination half-life (t_1/2β of 0.42 ± 0.08 h and 0.42 ± 0.09 h, respectively. The volume of distribution (V_d) was low, values of 0.20 ± 0.06 L/kg and 0.22 ± 0.06 L/kg being obtained for R(-) and S(+)KTP, respectively. Body clearance (CI₈) was high, correlating with the short elimination half-life, 0.3 3 ± 0.03 L/kg/h [R(-)KTP] and 0.32 ± 0.04 L/kg/h [S(+)-KTP]. KTP pharmacodynamics was evaluated by determining the effects on serum thromboxane (TxB₂), exudate prostaglandin (PGE₂), leukotriene (LTB₄) and β-glucuronidase (β-glu) and bradykinin (BK)-induced oedematous swelling. Effect-concentration inter-relationships were analysed by PK/PD modelling. KTP did not affect exudate LTB₄, but inhibition of the other variables was statistically significant. The mean EC₅₀ values for inhibition of serum TxB₂, exudate PGE₂ and β-glu and BK-induced swelling were 0.118, 0.086, 0.06 and 0.00029 μg/mL, respectively. These data indicate that KTP exerted an inhibitory action, not only as expected, on eicosanoid (TxB₂ and PGE₂) synthesis but also on exudate β-glu and BK-induced oedema. The EC₅₀ values for these actions indicate that they are likely to contribute to the overall anti-inflammatory effects of KTP in calves. However, claims that KTP inhibits 5-lipoxygenase and thereby blocks the production of inflammatory mediators such as LTB₄ were not substantiated. PK/PD modelling has proved to be a useful tool for analysing the in vivo pharmacodynamics of KTP and for providing new approaches to elucidating its mechanism(s) of action.     

Oral administration of tepoxalin in the horse: a PK/PD study

Tepoxalin is a non-steroidal anti-inflammatory drug with analgesic, anti-inflammatory, and antipyretic properties and has been recently introduced into veterinary medicine. The aim of this study was to evaluate the pharmacokinetic/pharmacodynamic (PK/PD) profile of tepoxalin to assess whether it would be suitable for clinical use in horses. Six female fasting/fed horses were given 10mg/kg tepoxalin orally in a cross-over study. After administration, tepoxalin underwent rapid and extensive hydrolytic conversion to its carboxylic acid metabolite RWJ-20142. In animals that had been fed, the plasma concentrations of tepoxalin were undetectable, whereas in fasting animals they were close to the limit of quantification of the method. No differences between the fasting/fed groups in RWJ-20142 plasma concentrations were shown. Tepoxalin showed a strong and long-lasting ex vivo inhibitory activity against cyclooxygenase (COX)-1, mainly due to its main metabolite RWJ-20142. Tepoxalin and RWJ-20142 do not seem to possess either COX-2 or 5-lipoxygenase inhibitory activity in the horse. These features suggest that the drug is a selective COX-1 inhibitor in horses, with no significant anti-inflammatory activity. Thus, its long term use in equine practice could be of concern.     

猪圆环病毒1型阴性的PK-15细胞株的筛选

PK15是猪肾传代细胞系,其父母代源自1955年美国Stice提供的成年猪肾细胞PK2a,后被美国ATCC收藏(收藏号为ATCCCCL33)[1].PK15细胞现已广泛应用于猪瘟病毒、猪伪狂犬病病毒、猪细小病毒等的分离、体外增殖以及多种兽用疫苗的生产.