牛GM-CSF与金黄色葡萄球菌黏附素FnBPB共表达质粒的构建与原核表达

采用PCR方法对牛粒细胞-巨噬细胞集落刺激因子(GM-CSF)和金黄色葡萄球菌FnBPB的D区进行特异性的扩增,并通过重叠延伸PCR扩增GM-CSF-FnBPB串联基因,构建了克隆质粒pMD19-GM-CSF-FnBPB。利用表达载体pET-32a(+)对该融合基因片段进行原核表达,SDS-PAGE分析表明,在1mmol/L IPTG诱导浓度下,在39ku处出现了与目的蛋白一致的外源蛋白带,Western blot分析表明,该蛋白具有反应原性,进而证明该融合基因成功在原核细胞中表达。     

Expression of receptor tyrosine kinases VEGFR-1 (FLT-1), VEGFR-2 (KDR), EGFR-1, PDGFRa and c-Met in canine primary brain tumours

Inhibition of tumour growth and angiogenesis by targeting key growth factor receptors is a promising therapeutic strategy for central nervous system tumours. Characterization of these growth factor receptors in canine primary brain tumours has not been done. Using quantitative real‐time TaqMan polymerase chain reaction (PCR), we evaluated the expression of messenger RNA (mRNA) for five tyrosine kinase growth factor receptors (vascular endothelial growth factor receptor [VEGFR]‐1, VEGFR‐2, endothelial growth factor receptor [EGFR]‐1, platelet‐derived growth factor receptor a [PDGFRa], and c‐Met) relative to normal cerebral cortex in 66 spontaneous canine primary brain tumours. Increased expression of VEGFR‐1 and VEGFR‐2 mRNA was greatest in grade IV astrocytomas (glioblastoma multiforme) and grade III (anaplastic) oligodendrogliomas. EGFR‐1 mRNA expression was more consistently increased than the other receptors in all tumour types, while increased PDGFRa mRNA expression was mostly restricted to oligodendrogliomas. The similarities in increased expression of these tyrosine kinase growth factor receptors in these canine tumours, as compared to data from their human counterparts, suggest that common molecular mechanisms may be present.     

Detection of Salmonella in Finishing Pigs on Farm and at Slaughter in Piedmont,Italy

Salmonella is one of the most common causes of human gastroenteritis often associated with pork consumption. The aims of this cross‐sectional study were to collect preliminary data on the presence of Salmonella enterica in pigs in Piedmont (Italy), through sampling on farm and at slaughter and to gather pilot data on serotypes and phagetypes present in the sampled area and distribution of anti‐microbial resistance among isolated strains. Salmonella was detected through culture and identified with Salmonella spp. and Salmonella Typhimurium PCR; positive samples were serotyped, phagetyped and tested for antibiotic susceptibility. Positive samples (from 9% of faeces up to 29% of tonsils) were found in 64% of the herds. Salmonella spp. was retrieved also from scalding water. Most of the isolates were Salmonella Derby, Salmonella Typhimurium and Salmonella 4,5,12:i:‐. The results of Salmonella Typhimurium specific PCR suggested that Salmonella 4,5,12:i:‐ might be unrecognized by serotyping. Anti‐microbial resistance was recorded in 75–100% of the isolates. Phagetyping allowed the identification of DT104B and DT46A strains. These results set the bases for further research studies that would aim to estimate the real herd prevalence in Piedmont and the diffusion of serotypes and anti‐microbial resistant strains within the same region.     

荧光定量PCR技术及其应用研究进展

荧光定量PCR是近年发展起来的一种新的实时定量检测特定核酸技术,它是核酸探针技术、荧光共振能量传递技术和PCR技术的有机结合,而荧光探针是荧光定量PCR的核心.荧光定量PCR具有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应争特点,是对原有PCR技术的革新,扩大了PCR的应用范围.论文综述了FQ-PCR技术的原理、FQ-PCR实时定量检测系统及其应用.     

紫花苜蓿PGIP同源基因克隆与序列核苷酸多态性分析

多聚半乳糖醛酸酶抑制蛋白(PGIP)是植物重要的防卫蛋白之一,了解PGIP基因的核苷酸序列是对其进行分子遗传学与分子生物学研究的前提与基础。本研究采用同源克隆法,从16个紫花苜蓿(Medicago sativa)品种的集群DNA中克隆了7个苜蓿PGIP基因的部分基因组编码序列,分别对应于蒺藜苜蓿(M. truncatula)7个不同的PGIP基因,并命名为MsPGIP5、MsPGIP6、MsPGIP7、MsPGIP8、MsPGIP9、MsPGIP10和MsPGIP11。其中,MsPGIP5、MsPGIP6、MsPGIP8、MsPGIP9和MsPGIP11的单核苷酸多态性(Single Nucleotide Polymorphism,SNP)位点间的平均距离分别为20、74、34、423和21 bp,差异较大。对MsPGIP5、MsPGIP6、MsPGIP8和MsPGIP11进行荧光标记PCR单链构象多态性(FPCR-SSCP)分析,结果表明,其等位基因数目与PCR片段长度的比值分别为20、56、22和19 bp,变化趋势与这4个基因SNP位点间的平均距离基本一致。比较而言,在发现的7个苜蓿PGIP基因中,MsPGIP9序列高度保守,MsPGIP5和MsPGIP11的变异较大,而MsPGIP6与MsPGIP8的变异中等。     

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。     

动物油脂中DNA的提取及牛、羊源性成分的PCR检测

用异硫氰酸胍法提取鱼油、加入牛肉DNA的鱼油、加入山羊肉DNA的互油和牛油中的DNA。18S rDNA片段以及牛、羊源性成分的PCR扩增结果表明，建立的动物油脂DNA提取方法是可行的。用建立的DNA提取方法提取3份送检鱼油的DNA，牛、羊源性成分的PCR检测结果均为阴性。     

鸡传染性贫血病病毒TaqMan实时荧光定量PCR检测方法的建立

根据鸡传染性贫血病病毒(chicken infectious anemia virus,CAV)基因组保守区域设计1对特异性引物和探针,通过优化反应条件,建立了一种快速检测CAV的TaqMan实时荧光定量PCR方法,同时验证其特异性、灵敏性和重复性.本试验建立的TaqMan实时荧光定量PCR方法灵敏度可达1.8×10¹拷贝/μL,远高于常规PCR方法;并与禽类其他病毒性疾病无交叉反应,具有高特异性.用分离的病毒人工感染1日龄SPF雏鸡,14日龄剖检,对感染鸡体内各器官的病毒分布及载量进行检测,结果表明,在脑、心脏、肝脏、脾脏、肺脏、肾脏、胸腺、法氏囊和血清中均可检测到病毒,肝脏、胸腺病毒载量明显高于其他组织.本研究建立的TaqMan实时荧光定量PCR方法特异性强、灵敏度高、重复性好,可同时检测大量临床样品,适用于CAV的诊断与流行病学分析.     

Unveiling patterns of viral pathogen infection in free-ranging carnivores of northern Portugal using a complementary methodological approach

Pathogen surveillance in free-ranging carnivores presents challenges due to their low densitie and secretive nature. We combined molecular and serological assays to investigate infections by viral pathogens (Canine parvovirus (CPV), Canine distemper virus (CDV) and Canine coronavirus (CCoV)) in Portuguese carnivores (Canis lupus, Vulpes vulpes, Lutra lutra, Martes foina, M. martes, Meles meles, and Genetta genetta) over a period of 16 years. Additionally we explored spatio-temporal patterns of virus occurrence in Canis lupus. Our study identified CPV DNA in all carnivore species with an overall prevalence of 91.9 %. CPV was detected in all sampled years and seasons in Canis lupus, supporting its enzootic nature. CDV RNA was mainly detected in the Canidae family, with viral nucleic acid recorded between 2005 and 2008 with a peak prevalence of 67 % among the wolf population, followed by a sharp decline, suggesting an epizootic behaviour of the virus. Antibodies show that mustelids and viverrids were often exposed to CDV. CCoV was first recorded by molecular methods in wolf samples in 2002, remaining in the wolf populations with marked fluctuations over time. The dual serological and molecular approach provided important epidemiological data on pathogens of wild carnivores in Portugal. These programmes should also include monitoring of other potential reservoir hosts such as domestic cats and dogs.