猪繁殖与呼吸综合征病毒GP5蛋白分段表达及其生物学功能的鉴定

为进一步研究PRRSV GP5蛋白的生物学功能,本研究将已分离的PRRSV北美洲型野毒株TA-12的GP5基因分为4段(79 bp～207bp、133bp～330bp、229bp～492bp和382bp～603bp)分别克隆于pGEX-6p-1中,并转化E.coli BL21(Rosetta)细胞进行诱导表达.表达蛋白经纯化后,以间接荧光方法(IFA)检测它们与Marc-145细胞的相互作用.IFA检测结果表明GST-GP5-1和GST-GP5-2融合蛋白均能够与Marc-145细胞结合,而且其结合位点在27aa～110aa区域.本实验为进一步研究GP5蛋白生物学功能提供试验依据.     

高致病性猪繁殖与呼吸综合征病毒HuN4株及其致弱过程中的不同代次病毒的分子特征分析

为了追踪高致病性猪繁殖与呼吸综合征病毒（Highly pathogenic porcine reproductive and respiratory syn-drome virus,HP-PRRSV）毒力逐渐减弱的分子轨迹,我们以HP-PRRSV亲本毒HuN4株及其系列传代致弱毒株为研究对象,对HuN4株及其不同代次毒株其全基因序列进行了序列比较分析。结果显示由高致病性的亲本毒HuN4株到无致病性的F112代的疫苗毒,共有65个核苷酸发生突变,其中导致41个氨基酸发生突变,这些突变的氨基酸分散性的存在于不同基因区域。与已经报道的高致病性JXA1株及其致弱毒株JXA80株相比较,可以看到两个强毒株毒力致弱的演变轨迹并不完全相同,二者之间不仅氨基酸突变数量不一致,而且突变氨基酸的位置也不完全相同,但其共同特点是由强毒株到弱毒株的变化过程中,突变率较高的结构蛋白相一致,突变率较高的非结构蛋白基本一致。分析结果提示我们决定高HP-PRRSV毒力强弱的因素可能不是由单个氨基酸的突变来决定的,推测是由一个基因或多个基因或多种因素共同造成的,同时也使我们认识到PRRSV的非结构蛋白区在病毒毒力变化中可能发挥重要作用。     

从鸭腹水综合征病鸭体内分离鉴定到血清1型鸭肝炎病毒

2011年4月份,河北省某地一些鸭场饲养的20～45日龄的樱桃谷肉鸭发生了以腹腔中充满大量清亮、茶色或啤酒样腹水为主要病变特征的疾病,即鸭腹水综合征。采集两个不同日龄的病死鸭肝脏样品,接种SPF鸭胚进行病毒分离,获得两个病毒分离物（HB01和HB02）。这两个病毒分离物对鸡、鸭和鹅的红细胞均无血凝活性。RT-PCR或PCR检测表明,HB01和HB02中鸭肝炎病毒检测为阳性,而鸭呼肠孤病毒、鸭圆环病毒、鸭黄病毒等检测均为阴性。将HB02接种5日龄SPF鸭,致死率为40%;对20日龄的鸭不致死。HB02分离物中鸭肝炎病毒的序列分析表明其与鸭肝炎病毒NA株及弱毒疫苗株C80和F64遗传距离较近。根据试验结果,推测鸭肝炎病毒可能是诱发本次鸭腹水综合症的因素之一。     

静脉注射纤维素微粒诱发肉鸡腹水综合征模型的建立

通过翅静脉注射CM-32纤维素微粒,观察血清白蛋白(ALB)、总蛋白(TP)、血细胞压积(PCV)、血红蛋白(Hb)、腹水等方面的变化,探讨建立肉鸡腹水综合征(AS)模型的可行性。试验分为2组,试验组在20日龄时注射0.3 mL纤维素悬液,对照组注射等量生理盐水。饲养期间统计临床型AS的发病率,并分别于21、28、35、42日龄从各组随机抽样,测定右心/全心重量比值(RV/TV),检查ALB、TP、PCV、Hb、腹水液等。结果试验组AS发病率(12%)显著高于对照组(3.3%),RV/TV值显著增加。纤维素微粒阻塞在肺血管,引起明显的炎性细胞聚集和肉芽结缔组织增生。试验组血清ALB、TP在42日龄明显低于对照组,PCV在28、35日龄时显著高于对照组(P<0.05),Hb在35日龄时显著高于对照组(P<0.05),其腹水液为典型的漏出液,表明翅静脉注射CM-32纤维素微粒成功诱发了AS。     

ORF2基因缺失的PCV2的构建与部分生物学特性

设计了1对特异性引物,从疑似PMWS（断乳仔猪多系统衰竭综合征）的病料中经PCR直接扩增出PCV2全基因组,再与载体pMD18-T Simple连接后形成重组质粒pMD18-T Simple-PCV2（命名为P-S-PCV2）。用ORF2特异的限制性内切酶EcoRⅠ对P-S-PCV2进行酶切、补平连接反应,构建了ORF2基因缺失突变的重组质粒（命名为P-S-PCV2-B）。用SacⅡ酶对基因缺失突变的重组质粒进行酶切,获得的线性化的基因突变PCV2基因组在体外进行自身环化,形成了相应的基因缺失病毒粒子（命名为PCV2-B）。用PCV2-B缺失突变株进行了细胞转染、动物致病性和免疫原性、T淋巴细胞亚群的动态变化等部分生物学特性的研究,结果显示,PCV2-B转染IBRS-2细胞后,电镜观察可见病毒颗粒,PCR-RFLP检测有突变株生长。PCV2-B接种仔猪后无临床典型大体病变,PCR-RFLP检测淋巴结中有PCV-B突变病毒的感染。PCV2-B免疫仔猪后抗体没有升高;CD3＋和CD4＋整体水平下降,第2周与对照组差异显著;CD8＋与对照组差异不显著。结果表明,ORF2基因缺失突变的PCV2仍具有复制感染能力,免疫原性减弱,细胞免疫被部分激活。     

规模猪场PRRS风险因素分析

猪繁殖与呼吸综合征造成养猪产业巨大经济损失,预防和消除该病已成为世界性难题。本文根据猪繁殖与呼吸综合征流行病学特性,结合规模化猪场生产管理实际,采用风险列举法对规模猪场PRRS传入和场内传播流行的风险因素进行了系统分析。提出规模猪场PRRS风险因素主要包括猪场选址、用品和工具进入、员工和访客进入、活猪运输、饲料运输、死猪处理、粪便管理、猪舍条件、生物传播媒介、引种等外部风险因素和PRRSV状态、管理措施、猪只流动、设施条件、协同病原、猪舍布局等内部风险因素。这为进一步开展规模猪场PRRS风险评估研究,制订科学有效的PRRS风险管理措施奠定了良好基础。     

Effects of oxidative stress on high-fat,high-salt diet and sucrose solution-induced metabolic syndrome in nephropathy rats

AIM: To establish a suitable animal model of nephropathy associated with metabolic syndrome (MS) induced by abnormal diet, and to investigate the effects of oxidative stress on renal damage in MS rats. METHODS: Normal 7-week-old male SD rats were randomly divided into 2 groups.The animals were fed with normal chow (control group, n=10) or high-fat and high-salt diet plus 20% sucrose solution (MS model group, n=10) for 20 weeks. Systolic blood pressure (SBP) was measured monthly. The levels of blood glucose, serum and urinary creatinine (Cr), total cholesterol (TC), triglycerides (TG), fasting insulin (FIns), urinary protein, urinary albumin and urinary sodium were determined. Insulin resistance (HOMA-IR), creatinine clearance (Ccr), urinary protein excretion (UPE), urinary albumin excretion (UAE) and urinary sodium excretion (USE) were calculated. Renal total-antioxidant capacity (T-AOC), inhibiting superoxide anion capacity (ISAC), malondialdehyde (MDA) content, and antioxidant enzyme activity were measured. Renal protein expression of Cu/Zn-SOD, NADPH oxidase subunit p47^phox and p22^phox was detected by Western blotting. In addition, pathological changes of the kidney were observed with PAS and Masson staining,and degree of glomerulosclerosis (GS) and tubulointerstitial injury was evaluated. RESULTS: Compared with control rats, SBP, TC, TG, FIns, USE and UAE were increased in MS rats. Furthermore, the MS rats showed a significant elevation of renal MDA content, p47^phox protein expression and GS score, and reduction of T-AOC, ISAC, SOD activity, and Cu/Zn-SOD protein expression in the kidney. CONCLUSION: SD rats fed with abnormal diet produce a suitable animal model of MS nephropathy that mimics the major features of human MS. Oxidative stress caused by up-regulation of NADPH oxidase expression and down-regulation of SOD expression may be one of the mechanisms leading to MS renal damage.     

鸢尾的花部结构及繁育系统特征

 对鸢尾（Iris tectorum）居群的花部特征、授粉特性、繁育系统及访花昆虫行为等进行了研究。在自然条件下,鸢尾4—6月开花,群体花期约60 d,群体盛花期约30 d,集中在4月20日—5月20日,单株花期10 ~ 30 d,单花花期2 d。雄蕊短于花柱,花药紧贴花柱外侧,花药与柱头相差约3 mm,在花朵开放的整个过程中雌、雄蕊的相对位置不变。杂交指数OCI为4,P/O（花粉/胚珠比）为234.4,结合坐果率判断其繁育系统属于异交,部分自交亲和,需要传粉者。鸢尾高度特化的花部特征与黄胸木蜂（Xylocopa appendiculata）和长木蜂（Xylocopa tranquebarorum）的传粉行为相适应。人工授粉结籽率高于自然传粉,证实鸢尾自然条件下的有性繁殖系统受到花粉限制和缺少传粉昆虫的影响。     

Effects of recruitment maneuvers on oxygen metabolism and hemodynamics in canine models of extrapulmonary and pulmonary ARDS

AIM: To determine the effects of recruitment maneuver(RM) strategy on oxygen metabolism and hemodynamics in canine models of extrapulmonary acut respiratory distress syndrome(ARDSexp) and pulmonary acute respiratory distress syndrome(ARDSp). METHODS: Twelve healthy canines were randomly divided into ARDSexp group and ARDSp group with 6 dogs each. To induce lung injury, the ARDSexp dogs were injected with oleic acid through femoral vein, and hydrochloric acid were administered by lung lavage to the ARDSp dogs. Pressure control ventilation (PCV) with lung protective ventilation strategy (LPVS) was employed to RM. P_high was set to upper inflection point (UIP), and P_low was set to lower inflection point (LIP)+2 cmH₂O.The duration of RM was 60 s and followed by baseline ventilatory settings. The indexes of oxygen metabolism and hemodynamics were measured before and after RM. RESULTS: After RM, arterial partial pressure of oxygen (PaO₂), venous partial pressure of oxygen (PvO₂), mixed venous oxygen saturation (SvO₂) and oxygen delivery (DO₂) obviously increased, and oxygen extraction ratio (ERO₂) gradually decreased in both groups. PaO₂, PvO₂, SvO₂ and DO₂ in ARDSexp group were significantly higher than those in ARDSp group, and ERO₂ in ARDSexp group were significantly lower than that in ARDSp group. Although mean pulmonary arterial pressure (MPAP), central venous pressure (CVP) and pulmonary artery wedge pressure (PAWP) significantly increased and mean arterial pressure (MAP) and cardiac index (CI) obviously decreased in both groups during RM, they reversed rapidly after RM. During RM, MPAP, CVP and PAWP were significantly higher, and MAP and CI were significantly lower in ARDSp group than those in ARDSexp group. CONCLUSION: RM increases oxygen delivery and improves tissue anoxia, and the better effects are obtained in ARDSexp dogs than those in ARDSp dogs. The effects of RM on hemodynamics are temporary and the ill effects are worse in ARDSp dogs than those in ARDSexp dogs.     

Role of 78 kD glucose-regulated protein in development of hepatopulmonary syndrome induced by multiple pathogenic factors

AIM: To explore the role of 78 kD glucose-regulated protein (GRP78) in the development of hepatopulmonary syndrome (HPS) in rats and the relation to intestinal endotoxemia (IETM). METHODS: The experimental animals were randomly divided into HPS groups of the 4th week, the 6th week and the 8th week. Normal control groups at the corresponding time points were also set up. The Wistar rat model of HPS produced in the process of liver cirrhosis was induced by employing multiple pathogenic factors to the animals. The rats in normal control group were designed by feeding with standard diet and tap water. Histopathological changes of the lung and liver were observed under microscope with the staining of hematoxylin and eosin (HE). The concentrations of alanine amino transferase (ALT), endotoxin and tumor necrosis factor α (TNF-α) in plasma, the contents of TNF-α and malondialdehyde (MDA) in the lung tissues were detected. The expression of GRP78 at mRNA and protein levels in the lungs was measured by the methods of RT-PCR and Western blotting, respectively. RESULTS: The level of endotoxin in plasma was gradually increased with the HPS development. The expression of GRP78 at mRNA and protein levels was also gradually increased with the HPS development and was significant at every time point. The endotoxin in plasma was positively correlated with the expression of GRP78 protein in the lung tissues of the rats with HPS. With the HPS development, the levels of ALT and TNF-α in plasma and the contents of TNF-α and MDA in the lung tissues were gradually increased. The content of endotoxin in plasma and the protein expression of GRP78 in the lung tissues were positively correlated with the contents of TNF-α in plasma and TNF-α and MDA in the lung tissues. The contents of TNF-α in plasma and GRP78 at mRNA and protein levels and TNF-α in the lung tissues were higher in the rats with HPS at every time point than those in normal control group. At the 6th week and the 8th week, the contents of endotoxin and ALT in plasma and MDA in the lung tissues of the rats with HPS were significantly higher than those in normal control group. CONCLUSION: IETM originated from the liver cirrhosis acts as a critical stressor of endoplasmic reticulum (ER) stress and activates ER stress in the lung by oxidative stress, resulting in increased expression of GRP78. Therefore,the increased expression of GRP78 induced by ER stress may play an important role in the development of HPS in rats.