鸡大肠杆菌活的非可培养状态发生相关基因克隆与分析

本研究旨在从分子水平探究细菌活的非可培养状态(VBNC)的发生机制。应用冰乙酸和4℃联合诱导条件,使鸡大肠杆菌进入VBNC状态,并利用mRNA差异显示技术(DDRT-PCR)获得VBNC相关基因。结果表明,从VBNC大肠杆菌中筛选得到的3个差异片段与大肠杆菌23S核糖体RNA基因序列具有较高的核苷酸同源性,分别为98%、98%和99%,而氨基酸的同源性也均在97%以上,表明这3个序列是大肠杆菌23S核糖体rRNA基因的部分序列,同时也是与大肠杆菌VBNC状态发生密切相关的基因。由此推知,当正常大肠杆菌在未暴露任何压力下时,其转录水平较低,特别是23S rRNA的某一(些)基因不显示或受到强烈抑制。当进入VBNC状态后,面临生存压力时,这一(些)基因转录水平明显强于正常状态,而核糖体作为蛋白质合成的主要结构与场所,其某些基因也将积极参与新蛋白质的生物合成。     

SOE PCR重构SLA-Ⅰ复合体研究

利用剪接重叠延伸PCR(SOE PCR)技术,中间加一富含甘氨酸/丝氨酸的linker(G4S)3,将SLA-Ⅰ重链基因SLA-2和轻链基因β2m连接,形成复合体基因链SLA-2-(G4S)3-β2m,然后将复合体基因链克隆入p2X表达质粒,导入受体菌TB1并进行表达。结果显示,利用SOE PCR技术成功得到了复合体基因链SLA-2-(G4S)3-β2m,并且克隆入p2X载体。SDS-PAGE检测表明,复合体基因导入宿主菌后获得了融合蛋白MBP-SLA-2-(G4S)3-β2m,大小为84.1 ku。试验结果为今后在体外进行相关基因重组表达提供参考。     

转基因牛多重PCR检测方法的建立

为建立一种快速检测转基因牛的多重PCR方法,针对牛线粒体16 S rRNA基因(BOS mtD-NA16 S rRNA,BOS)、转基因动物常用标记基因新霉素磷酸转移酶基因(NPT Ⅱ)、人乳铁蛋白编码基因(human laetoferrin gene,hLF)、人-α-乳清蛋白编码基因(human α-lactalb...     

应用逆转录套式PCR检测新城疫病毒核酸

选择DNV融合蛋白保守的编码区域,设计并合成了一对外引物和一对内引物,建立并优化了检测新城疫病毒核酸的逆转录套式PCRI地,通过检测NDV感染的实验客观存在病料和临床病料,结果表明,逆转录套式PCR法最低能鉴别出约0.3pg的NDV RNA,攻毒后第8天还能从非免疫鸡和SPF鸡泄殖腔拭子中检出DNV,第8天非免疫鸡泄殖腔拭子中NDV的最大检出率为5/10,第8天SPF鸡泄殖腔拭子中NDV的最大检出率为6/10,对非免疫鸡和SPF鸡的泄殖腔中NDV最佳检出时间均在攻毒后第5天。逆转录套式PCRY应运地临床样品中DNV的最大检出率为6/7,经核酸杂交验证,该法具有很高的特异性和敏感性,也比较简便快速,为从分子水平探讨NDV的发病机理、临床早期快速诊断提供了新的研究手段。     

Identification of lactic acid bacteria in the feces of dairy cows fed whole crop maize silage to assess the survival of silage bacteria in the gut

In order to assess the survival of lactic acid bacteria (LAB) in whole crop maize silage in the gut of dairy cows, one representative silage sample and three different feces samples were collected from dairy cows on three dairy farms in Hua Bei, China and three dairy farms in Kyushu, Japan. The composition of the bacterial community was examined by denaturing gradient gel electrophoresis and quantitative polymerase chain reaction. Lactobacillus acetotolerans was detected in all bunker‐made maize silage samples, regardless of the dairy farm or sampling region from which they were sourced. A total of eight LAB species were detected in the maize silage samples, of which three (L. acetotolerans, L. pontis and L. casei) appeared to survive digestion. The populations of L. acetotolerans in silage and feces were 10^6–7 and 10^3–4 copies/g, respectively, indicating that, even for the LAB species showing potential survival in the gut, competition in this niche may be harsh and the population may substantially decrease during the digestion process. It may be difficult for silage LAB to survive in the gut of silage‐fed dairy cows, because marked decrease in population can take place during the digestion process, even for surviving species.     

高分辨率熔解曲线分析法检测牦牛脂蛋白脂酶外显子7的多态性及与生长性状相关性分析

脂蛋白脂酶(lipoprotein lipase,LPL)是一个分子质量为54 kb的糖蛋白,其活性受营养因素、激素水平等调节,因牦牛生存环境的特殊性,试验拟从分子水平研究天峻县牦牛脂蛋白脂酶外显子7(LPL-Exon7)基因的多态性,并与生长发育性状进行关联分析,从而为牦牛营养调控、品种资源保护和遗传育种提供科学理论依据。试验采集青海省海西州天峻县牦牛抗凝血106份,提取血样DNA,并利用PCR技术和高分辨率熔解曲线分析法对候选基因LPL-Exon7进行多态性分析,并于采血的同时对牦牛体重、体斜长、体高、胸围和管围等生长性状指标进行了相关性和遗传效应分析。结果表明,高原牦牛LPL-Exon7基因在高原牦牛和野血牦牛中都表现为多态,存在3种基因型,即AA、AB和BB,由1对等位基因A和B控制,A等位基因均为优势等位基因。经χ²检验,符合哈迪—温伯格(Hardy-Weinberg)定律,处于平衡状态(P>0.05)。该位点在高原牦牛和野血牦牛群体中公、母牦牛的多态信息含量为0.3588和0.2103、0.3219和0.3343,高原牦牛母牛的多态信息含量PIC<0.25,处于低度多态,高原牦牛公牛和野血牦牛公、母牛的多态信息含量均在0.250.05)。因此,初步推断牦牛LPL-Exon7可以作为牦牛标记辅助选择的遗传标记之一。     

First report of Wolbachia from field populations of Culex mosquitoes in south-western Nigeria

Recent reports on finding Wolbachia-strain infections in field mosquito species in some West African countries and the potential for developing these as disease vector biocontrol tools have prompted a search for Wolbachia in mosquitoes within the study area. Using a completely randomised design, mosquito traps were set at different locations in a rural and an urbanised community. One hundred and eighty (180) mosquitoes were trapped and pooled on the basis of genus, sex and site of collection, because there have been no earlier reports of Wolbachia isolated from Nigeria. Twenty pools, made up of not more than ten mosquitoes per pool, were homogenised and analysed for Wolbachia-specific DNA. Mosquitoes were trapped within Ede (urbanised community) and Akoda (rural community). Genomic DNA was extracted from trapped mosquito samples and used as a template in a PCR reaction. The Wolbachia sp. specific 16S rRNA gene was amplified, sequence analysis of PCR products was performed and a chromatogram of the sequence was subjected to Basic Local Alignment Search Tool analysis to identify the Wolbachia sp. This sequence was subsequently submitted to GenBank with accession number MK127541. The first evidence of the presence of the endosymbiont, Wolbachia in field-caught mosquitoes is hereby documented. The homology of this strain of Wolbachia bears similarities to those reported recently from other parts of West Africa and forms a single clade with a Wolbachia sp. from Mali, with a strong bootstrap support of 99%. This finding of a Wolbachia strain in mosquitoes at Ede could form the basis for more searches for diverse strains of Wolbachia in Nigeria.     

Expression of RANTES mRNA in skin lesions of feline eosinophilic plaque

Abstract One of the mechanisms of eosinophil infiltration is its induction by chemoattractants such as regulated upon activation, normal T-expressed and secreted (RANTES) which is a cysteine–cysteine chemokine that mediates chemotaxis and activation of eosinophils in humans and mice. Skin lesions of feline eosinophilic plaque are characterized by a predominant infiltration of eosinophils. The mechanism(s) of eosinophilic infiltration in the skin and/or mucosa of cats is unknown. It is possible that RANTES is involved. To investigate the presence of RANTES in the skin of cats with eosinophilic plaques and nonaffected skin, we cloned and sequenced the full-length feline RANTES cDNA gene, in order to determine whether it is present in the skin of cats with eosinophilic plaques and/or if it is present in normal adjacent skin. We were able to document the the expression of RANTES mRNAs in skin with feline eosinophilic plaque as well as in normal cat skin. The full-length cDNA sequence of the RANTES gene (742 bp) contained a single open reading frame of 276 bp encoding a protein of 92 amino acids. The amino acid sequence of feline RANTES shared 67 and 74% sequence identity with that of bovine and mouse RANTES genes, respectively. RT–PCR analysis on RANTES mRNA in the skin of cats with eosinophilic plaque revealed that its expression was higher in the eosinophilic plaque skin lesions than in the normal skin. The result suggested that RANTES might play a role to induce eosinophil infiltration in feline eosinophilic plaque lesions.     

用PCR方法检测猪圆环病毒Ⅱ型

建立了一种能特异性地扩增猪圆环病毒 型 (porcine circovirus type 2 ,PCV- 2 )核酸片段的 PCR方法 ,从疑似断奶猪多系统衰竭综合征 (PMWS)的感染病例的淋巴结和脾脏中扩增出了预期长度的 PCV- 2 DNA片段 ,用限制性内切酶 Eco R 酶切鉴定了 PCR产物的特异性。 PCR产物的核苷酸序列分析表明 ,与已报道的 PCV- 2 (Gen Bank Ac-cession num ber AF0 2 72 17)相关区域的核苷酸序列同源性均大于 96 %。首次证实了我国猪群中存在 PCV- 2感染的PMWS。     

应用PCR检测鸭肠炎病毒弱毒在鸡胚体内的分布

根据GenBank已发表的鸭肠炎病毒（DEV）UL6基因的部分核苷酸序列，设计并合成一对引物．以鸭肠炎病毒中国商品化疫苗毒蚀斑纯化株（DEV Clone-03）DNA为模板，经PCR扩增出预期516bp的目的片段。将此片段插入克隆载体质粒pMD18-T，并进行序列测定，序列比较结果显示，此段序列与参考序列完全一致。用PCR检测DEV Clone-03在鸡胚体内的分布情况显示，接毒死亡鸡胚的尿囊膜、心、肝、脾、肺、肾和肌肉皮肤内均存在病毒。PCR敏感试验表明此方法可以检测到100μL尿囊液中提取的10^-3稀释的病毒DNA，相当于0．2个EID50病毒量的DNA。