猪繁殖与呼吸综合征病毒河北地方株ORF6基因的克隆及原核表达

从河北沧州分离到一株疑似猪繁殖与呼吸综合征病毒,接种Marc-145细胞,经4代盲传,出现细胞病变,经鉴定为PRRSV,命名为HB-3(cz)株。根据GenBank公布的PRRSV JXA1株ORF6基因的核苷酸序列,设计并合成一对特异性引物(P1/P2),用RT-PCR方法扩增完整ORF6基因,将扩增产物连接到pGM-T载体并转化克隆菌,阳性重组质粒PGM-M进行序列测定与分析。后将克隆质粒PGM-M双酶切后连接原核表达载体pET-32a(+),在Rosseta-DE3中成功获得表达,经Western-blotting分析表明,表达蛋白与阳性血清发生特异性反应。     

Antigen-nonspecific macrophage factors modulating the antibody response in vitro

Antibody response to an antigen involves the co-operation between three types of cells: macrophages, T cells and B cells. The cognate interactions between these cells play a fundamental role in the expression of a specific antibody response, but the last is modulated by antigen-nonspecific soluble factors produced either by macrophages or by T cells. Macrophages elaborate a spectrum of molecules modulating the function of lymphoid cells; among them are IL1 and prostaglandins of the E series, which are respectively enhancer and inhibitor of the antibody response in vitro. These molecules alter T cell and B cell activities through different mechanisms involving activation or inhibition of IL2 production, or alteration of cells surface antigens. However, the cellular events following the fixation of soluble factor on its receptors are not known.     

Effect of the β‐carotene oxygenase 2 genotype on the content of carotenoids,retinol and α‐tocopherol in the liver,fat and milk of rabbit does,reproduction parameters and kitten growth

Mutations in the β‐carotene oxygenase 2 (BCO2) gene can impair the function of the enzyme that breaks down carotenoids. As a result, gradual accumulation of unoxidized carotenoids in animal tissues gives them a yellow colour. The aim of the study was to determine the content of carotenoids, retinol and α‐tocopherol in the liver, fat and milk of rabbit does with three different genotypes determined by AAT‐deletion mutation at codon 248 of the BCO2 gene and to find out whether differences in the concentrations of the above compounds in the tissues and milk of the does affect reproduction parameters and the rearing rate of kittens. The experimental materials comprised 36 does, 12 of each genotype of the BCO2 gene, with their litters. Females with their litters were placed in individual cages, on deep litter. Between days 7 and 13 of lactation, samples of milk were collected from the does. The kittens stayed with their mothers until 35 days of age. After weaning, the does were sacrificed. Tissue samples of liver and perirenal fat were collected for chemical analyses. Additionally, based on samples taken from one female, RNA expression levels were determined from the mammary gland and liver, adipose tissue and skin. It was found that homozygous does with deletion at codon 248 of the BCO2 gene were characterized by considerably higher concentrations of xanthophylls and beta‐carotene in the liver, adipose tissue and milk than does with the remaining genotypes. However, the differences in the content of the above compounds in milk had no influence on litter weight or the number and rearing rate of kittens. Additionally, RNA expression of the BCO2 gene was found in the mammary tissue of lactating doe and its level was similar to those noted in the liver and adipose tissue.     

山羊碱性氨基酸转运载体基因SLC3A1cDNA序列克隆及表达

试验旨在克隆山羊碱性氨基酸转运载体基因SLC3A1 cDNA序列,探究其表达的组织特异性及其在小肠中的表达发育模式。参考GenBank已发表的绵羊、牛SLC3A1基因mRNA序列设计引物,克隆山羊SLC3A1基因的cDNA序列,采用实时荧光定量PCR的方法分析其在1日龄山羊11种组织及其在1日龄、6月龄、8月龄、10月龄、12月龄山羊十二指肠、空肠、回肠中的mRNA表达情况。结果表明,成功克隆得到了山羊SLC3A1基因cDNA序列,其与绵羊、牛、野猪、人、小鼠、大鼠的同源性分别为99%、97%、88%、86%、80%和79%。SLC3A1基因转录表达有明显的组织特异性,其在1日龄山羊肾脏、回肠、空肠、结肠中表达量依次降低(P<0.05),其他组织中表达量很低。同一月龄山羊,SLC3A1基因在不同肠段的表达量数值上回肠>空肠>十二指肠。SLC3A1基因在不同月龄山羊十二指肠表达量以1日龄山羊为最高(P<0.05),6~12月龄山羊相同肠段之间表达量无显著差异(P>0.05);空肠表达量随山羊年龄的增加均呈现逐步降低的趋势;回肠表达量在各个月龄山羊之间差异不显著(P>0.05)。结果说明,山羊碱性氨基酸转运载体基因SLC3A1的主要表达部位在肾脏和肠道,推测b^0,+碱性氨基酸转运系统转运氨基酸的主要部位为肾脏和肠道。SLC3A1基因在山羊小肠受肠段和发育阶段的影响,具有不同的表达发育模式。     

microRNA-124-3p调控H1N1亚型猪流感病毒致小鼠肺损伤的研究

为研究microRNA-124-3p（miR-124-3p）对H1N1亚型猪流感病毒（swine influenza virus,SIV）感染小鼠所致肺损伤的调控作用,本试验构建miR-124-3p腺病毒表达载体,通过小鼠尾部静脉注射法构建miR-124-3p差异表达小鼠模型,试验分3组：过表达组、抑制组和对照组。48 h后,各组小鼠鼻腔接种H1N1亚型SIV,每只10⁵ EID₅₀（50 μL）。连续观察14 d,计算小鼠平均体重变化率、观察病理切片并测定相关炎症因子IL-1β、TNF-α和IL-6 mRNA相对表达量。结果显示,已成功将pre-miR序列及其sponge序列插入腺病毒的穿梭质粒,并将其共转染293A细胞。实时荧光定量PCR检测证实,与对照组相比,过表达组和抑制组小鼠黑色素瘤细胞miR-124-3p表达水平分别极显著升高（P<0.01）和显著降低（P<0.05）,表明成功构建腺病毒表达载体。过表达组、抑制组和对照组小鼠体重变化率分别为－5.5%、－12.4%和－8.6%。抑制组和对照组均可见肺泡壁增厚,其间有多量淋巴细胞浸润,部分肺泡内出现纤维蛋白渗出,且抑制组病理变化更为严重,肺泡中还有大量的红细胞浸润;而过表达组仅有少量的淋巴细胞浸润,肺脏组织较正常。与对照组相比,过表达组检测的炎症因子IL-1β、TNF-α和IL-6 mRNA表达水平均显著降低（P<0.05）;抑制组炎症相关炎症因子mRNA表达水平均显著升高（P<0.05）。本试验结果表明,miR-124-3p对H1N1亚型SIV感染小鼠所致的肺脏炎症因子的表达具有抑制作用,同时能减轻肺脏病理损伤。     

中能中蛋白水平下维生素A、D3对0～6周龄肉仔鸡生产性能的影响

采用3×3随机交叉试验设计,研究中能中蛋白水平下日粮VA、VD3对肉仔鸡生产性能的影响。1日龄AA肉仔鸡540只随机分为9组,每组3个重复,每重复20只鸡。3个VA添加水平为3000、6000、12000IU/kg;3个VD3添加水平为200、400、800IU/kg;0～3和4～6周龄基础日粮代谢能、粗蛋白水平分别为12.34MJ/kg,21%;12.76MJ/kg,19%。结果表明:①日粮VA水平对体增重、耗料量、料重比、存活率均无显著影响(P>0.05);②随VD3添加水平的增加,体增重、耗料量、存活率显著增加(P<0.05);③综合考虑生产性能和经济效益,认为0～3和4～6周龄肉仔鸡日粮VA添加量均以3000IU/kg较为适宜,VD3添加量均以800IU/kg较为适宜。     

钙和维生素D3对肉仔鸡肉品质和血液生化指标的影响

本试验旨在研究饲粮中添加高水平的钙和维生素D3对AA肉仔鸡肉品质和血液生化指标的影响。试验采用2×2完全随机因子设计,选用240只21日龄AA肉仔鸡,随机分成4个处理,每个处理6个重复,每个重复10只。试验期为28 d。玉米-豆粕型基础日粮中含钙0.90%、维生素D3 750 IU/kg,日粮中共设两个钙水平(0.9%,1.15%)和两个维生素D3水平(750、5000 IU/kg)。结果表明:(1)在AA肉仔鸡饲粮中单独添加1.15%的钙对肌肉嫩度影响显著(P<0.05),5000 IU/kg维生素D3对肌肉嫩度影响不显著(P>0.05),同时添加钙和维生素D3的互作效应对肌肉嫩度影响显著(P<0.05);(2)1.15%的钙显著降低胆固醇、甘油三酯、低密度脂蛋白的含量(P<0.05),显著升高了高密度脂蛋白的含量(P<0.05);(3)单独添加5000 IU/kg维生素D3显著降低了低密度脂蛋白的含量(P<0.05)。以上研究结果表明,玉米-豆粕型基础日粮中长期添加1.15%钙和5000IU/kg维生素D3能够减小肌肉剪切力,降低血液中总胆固醇和甘油三酯的含量,从而提高肉品质。     

中国对虾(Fenneropenaeus chinensis)“黄海3号”新品种的培育

2006年收集中国对虾“黄海1号”保种群以及海州湾、莱州湾2个野生群体为基础群体构建核心育种群,采用群体选育技术以仔虾Ⅰ期耐氨氮胁迫成活率和收获时对虾体重为选育指标,经过连续5代选育,培育出中国对虾“黄海3号”新品种。新品种耐氨氮胁迫能力强,仔虾Ⅰ期成活率较商品苗种提高21.2%,养殖成活率提高15.2%;生长速度快,收获对虾平均体重较商品苗种提高11.8%。AFLP技术分析获得5代选育群体的平均多态位点比例分别为42.28%、40.64%、40.32%、39.95%和38.05%。研究结果显示,随着选育世代的增加,选育群体的遗传多样性呈现下降趋势,但随着选育时间的延长,世代之间的分化逐渐降低,群体的遗传结构开始趋于稳定。     

乙酸钠对油酸诱导的BRL-3A细胞脂肪变性的影响及机制分析

通过向油酸诱导的大鼠肝成纤维细胞(BRL-3A)脂肪变性模型中添加不同浓度(2、4、8 mmol·L^-1)的乙酸钠,探讨其对脂肪变性细胞模型脂代谢的调控机理及细胞损伤的修复作用。试验方法：1)用不同浓度的油酸(0、0.03、0.06、0.12、0.24、0.48) mmol·L^-1刺激BRL-3A细胞24 h后,分别检测细胞相对活力、总脂滴面积、三酰甘油(TG)含量、天门冬氨酸氨基转移酶(AST)及丙氨酸氨基转移酶(ALT)活性,建立脂肪变性细胞模型;2)向BRL-3A细胞中添加不同浓度的乙酸钠,通过流式细胞术检测细胞凋亡率;3)用不同浓度的乙酸钠和0.12 mmol·L^-1油酸共同孵育BRL-3A细胞,试验分为4组,分别为油酸处理组、2 mmol·L^-1乙酸钠+油酸处理组、4 mmol·L^-1乙酸钠+油酸处理组和8 mmol·L^-1乙酸钠+油酸处理组,分别对细胞脂滴、TG含量、AST、ALT活性、AMPK信号通路蛋白以及脂代谢关键基因进行检测。结果显示:1)用0.12 mmol·L^-1油酸处理BRL-3A细胞24 h,成功建立BRL-3A细胞脂肪变性模型。2)不同浓度的乙酸钠对BRL-3A细胞凋亡率没有影响;3)4、8 mmol·L^-1乙酸钠处理脂肪变性细胞模型后,与油酸处理组相比,细胞总脂滴面积、每平方毫米脂滴数、TG含量、AST和ALT活性均显著(P<0.05)或极显著(P<0.01)下降,P-AMPK表达水平显著(P<0.05)或极显著(P<0.01)上升;脂合成代谢相关基因ACC、FAS以及SCD-1 mRNA表达水平均有一定程度下降;脂分解代谢相关基因CPT-1、CPT-2以及ACO mRNA表达水平均有一定程度上升。本研究表明,乙酸钠会通过AMPK通路激活脂分解代谢,减轻肝细胞脂质蓄积,并且对油酸诱导的BRL-3A细胞脂肪变性模型的损伤具有一定缓解作用。