培养液中铜水平对PK15细胞内铜离子浓度与CuZn-SOD活性的影响

在 PK15细胞无血清液体培养基中添加 0、7.8、15 .6、31.2、6 2 .5 μmol/L Cu,观察了培养液中不同铜水平对PK15细胞内铜离子浓度和 Cu Zn- SOD活性的影响及二者的相关性。结果表明 ,PK15细胞内铜离子浓度及 Cu Zn-SOD活性均随培养液中添加铜离子浓度升高和培养时间的延长而有不同程度的升高 ,其中以添加 Cu31.2 μmol/L组最高 (P<0 .0 5 )。PK15细胞内 Cu Zn- SOD活性与细胞内铜离子浓度和培养时间呈强正相关 (培养时间 :r=0 .873～0 .980 ,P<0 .0 5 ;铜离子浓度 :r=0 .90 0～ 0 .972 ,P<0 .0 5 )     

Susceptibility of various ceil culture systems to pseudorabies virus

A comparative study was carried out to determine the susceptibility of five different cell lines to pseudorabies virus (PRV), a herpes virus of pigs. The cell systems tested were swine testicle (ST), mink lung (ML), equine dermal (ED), porcine kidney (PK₁₅), and bovine turbinate (BT) cells. Virus titers obtained were 10^4.88, 10^4.38, 10^3.75, 10^2.63, and 10^0.25 for ML, ST, PK₁₅, BT and ED cells, respectively indicating that ML, ST, and PK₁₅ are optimal cell lines for the growth of PRV whereas BT and ED are not very sensitive.     

3种介导猪NR2F2基因转染PK15细胞方法的比较研究

试验旨在研究3种转染方法对猪肾上皮细胞（PK15）的转染效率,为以PK15细胞为模型研究外源基因的功能提供参考。本研究以PK15细胞为研究对象,用脂质体、电穿孔、慢病毒3种转染方法转染后,在荧光显微镜下观察绿色荧光蛋白的表达,采用实时荧光定量PCR检测EGFP和NR2F2基因的表达情况,采用CCK-8检测细胞存活率,进而比较3种方法的转染效果。结果显示,转染PK15细胞后,电穿孔和慢病毒方法转染效率极显著高于脂质体（P<0.01）,但电穿孔方法和慢病毒方法之间差异不显著（P>0.05）;EGFP和NR2F2基因的实时荧光定量PCR结果显示慢病毒方法效果最好,脂质体方法较差,与细胞转染效率基本一致;CCK-8结果显示,电穿孔转染后细胞存活率最低,极显著低于对照组（P<0.01）,脂质体方法显著低于对照组（P<0.05）,慢病毒方法与对照组间差异不显著（P>0.05）。综合考虑转染效率及细胞活性,本研究认为慢病毒转染方法最适合转染PK15细胞,为今后高效转染PK15细胞提供了理想的方法。     

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。     

饲料糖脂比对吉富罗非鱼生长、血液指标和肝脏糖代谢关键酶活性及基因表达的影响

为探讨糖脂比对吉富罗非鱼幼鱼生长性能、血液生化指标和肝脏糖代谢关键酶的影响,本实验设计了5种不同糖脂比(1.5、2.3、3.9、7.0、16.5)的等氮等能饲料,以吉富罗非鱼幼鱼为实验对象,进行了为期8周的饲养实验。结果显示,随饲料中糖脂比升高,吉富罗非鱼特定生长率(SGR)、增重率(WGR)和蛋白质效率(PER)先升高后降低,糖脂比为3.9和7.0时,WGR、SGR和PER最高。饵料系数(FCR)的变化趋势与此相反,在糖脂比为3.9和7.0组显著低于其他组。随着饲料糖脂比的增加,吉富罗非鱼全鱼粗脂肪含量逐渐下降,在糖脂比为16.5时达到最低水平,但仅与1.5组存在显著差异。血浆甘油三酯在糖脂比最高组也出现显著降低,血浆胆固醇同样在糖脂比最高组显著低于3.9组。然而血糖的变化趋势相反,当糖脂比为16.5时显著高于其他组。随着糖脂比的升高,吉富罗非鱼幼鱼肝脏中丙酮酸激酶(PK)的活性及mRNA表达量逐渐升高,而磷酸烯醇式丙酮酸羧激酶(PEPCK)的活性及mRNA表达水平则逐渐下降。根据二次回归模型得出,在等氮等能的饲料条件下,吉富罗非鱼幼鱼最适宜的糖水平和脂肪水平分别为49.69%和9.53%,其对应的糖脂比为8.4。     

PK15细胞全悬浮摇瓶培养工艺初探

PK15细胞是一种被广泛应用于兽用疫苗生产的细胞,然而现今生物制品行业快速发展,传统贴壁培养PK15细胞已经不能满足生产需要.目前通过全悬浮培养技术来培养细胞、病毒已经是发展趋势,但是由于生产用细胞多数为贴壁细胞,难以实现悬浮培养,所以,目前该项技术在疫苗生产上还未广泛应用,技术也还未成熟.故本文探讨PK15细胞在无血...     

Crop Nutrient Status and Nitrogen,Phosphorus, and Potassium Balances Obtained in Field Trials Evaluating Different Fertilizer Recommendation Systems on Various Soils and Crops in Hungary

Based on correlations calculated from the database of long-term fertilization experiments carried out in Hungary between 1960 and 2000, a new, cost-saving, and environmentally friendly fertilizer recommendation system was developed. The aim of the new system is to apply the lowest possible nitrogen (N)–phosphorus (P)–potassium (K) rates required to achieve good yields and maximum income per unit area. The World Phosphorus Institute (IMPHOS) agreed to finance a 3-year program to test various Hungarian fertilizer recommendation systems (the new RISSAC-RIA [Research for Soil Science and Agricultural Chemistry‐Research Institute for Agriculture] system, the Talajer?gazdálkodás integrated soil fertility management system, and the intensive MÉM NAK [Department of Plant Protection and Agricultural Chemistry of the Ministry of Food and Agriculture] system) when applied to three major crops grown on three characteristic Hungarian soils. The first five treatments in the experiment represented a classical deficiency experiment, aimed at checking the correctness of the NPK supply categories determined by the various systems. On all three soils and for all three crops the IMPHOS experiments confirmed the basic principles of the new cost-saving, environmentally friendly fertilizer recommendation system and the correctness of the methods used for calculating fertilizer rates. In this article, diagnostic plant analysis, soil analysis data, and NPK balances obtained in the different recommendations are discussed. In most cases the plant NPK contents of all the recommendations exceeded the lower limits of the good NPK supply category for wheat and barley shoot as well as for corn leaf. Crop NPK contents in Talajer?gazdálkodás and MÉM NAK treatments (with greater recommended NPK doses) were most cases better than in the RISSAC-RIA treatments; however, the better crop NPK status did not result in extra grain yield in the former treatments. Taking into account that similarly high yields were obtained using the RISSAC-RIA system and the Talajer?gazdálkodás integrated system with much smaller NPK doses than those recommended by the intensive MÉM NAK system, it was more advantageous from agronomic, environmental protection, and economic points of view. Differences in PK balances correlated well with the differences in the measured soil ammonium lactate (AL)–PK contents after the third year of the experiment.     

SINPV PK基因的部分序列分析

重组质粒ｐＰ１８ＰＨ３上含有ＳＩＮＰＶ的部分蛋白激酶基因。ＳＩＮＰＶ部分的蛋白激酶氨基酸序列与ＨａＮ－ＰＶ、ＨｚＮＰＶ、ＳｐｌｉＮＰＶ、ＡｆＮＰＶ、ＡｃＮＰＶ、ＬｄＮＰＶ蛋白激酶的氨基酸序列的同源性分别为４５％、８９％、３７％、４４％、３８％和３７％，其上含有蛋白激酶特征序列ＩＶＨＡＮＤＶＫＬＥＮＶＬ。     

Integration of pharmacokinetic–pharmacodynamic for dose optimization of doxycycline against Haemophilus parasuis in pigs

The aims of this study were to establish optimal doses of doxycycline (dox) against Haemophilus parasuis on the basis of pharmacokinetic–pharmacodynamic (PK‐PD) integration modeling. The infected model was established by intranasal inoculation of organism in pigs and confirmed by clinical signs, blood biochemistry, and microscopic examinations. The recommended dose (20 mg/kg b.w.) was administered in pigs through intramuscular routes for PK studies. The area under the concentration 0‐ to 24‐hr curve (AUC_0–24), elimination half‐life (T_½ke), and mean residence time (MRT) of dox in healthy and H. parasuis‐infected pigs were 55.51 ± 5.72 versus 57.10 ± 4.89 μg·hr/ml, 8.28 ± 0.91 versus 9.80 ± 2.38 hr, and 8.43 ± 0.27 versus 8.79 ± 0.18 hr, respectively. The minimal inhibitory concentration (MIC) of dox against 40 H. parasuis isolates was conducted through broth microdilution method, the corresponding MIC₅₀ and MIC₉₀ were 0.25 and 1 μg/ml, respectively. The Ex vivo growth inhibition data suggested that dox exhibited a concentration‐dependent killing mechanism. Based on the observed AUC_24 hr/MIC values by modeling PK‐PD data in H. parasuis‐infected pigs, the doses predicted to obtain bacteriostatic, bactericidal, and elimination effects for H. parasuis over 24 hr were 5.25, 8.55, and 10.37 mg/kg for the 50% target attainment rate (TAR), and 7.26, 13.82, and 18.17 mg/kg for 90% TAR, respectively. This study provided a more optimized alternative for clinical use and demonstrated that the dosage 20 mg/kg of dox by intramuscular administration could have an effective bactericidal activity against H. parasuis.     

OsMAPK7基因的克隆、序列分析及其植物表达载体的构建

以低温处理的耐盐水稻辽盐241植株叶片总RNA为模板,用OsCDPK7基因特异引物通过RT-PCR扩增出1 700 bp的片段,并将该片段克隆至pUC18上。序列分析结果表明,该序列与GenBank上的OsCDPK7基因序列相比,缺失了CDS序列中157￣234处的78个核苷酸,其编码的蛋白序列缺失了53￣78的26个氨基酸。但是缺失部分位于丝氨酸/苏氨酸蛋白激酶活性中心和钙结合基序等结构域之外,因此预计该基因产物应具备钙依赖的蛋白激酶活性。进一步将OsCDPK7基因分别克隆至植物表达载体卡盒pBE12和pB29A上,构建了分别由E12启动子、rd29A启动子调控的OsCDPK7基因植物表达载体pBC7E12和pBC729A。通过冻融法将重组质粒导入根癌农杆菌LBA4404中,为农杆菌介导法转化植物,分析OsCDPK7基因的功能奠定了基础。