Gene expression profile of HIRRV G and N protein gene vaccinated Japanese flounder, Paralichthys olivaceus during HIRRV infection

The glycoprotein (G protein) gene, but not the nucleocapsid protein (N protein) gene, of the hirame rhabdovirus (HIRRV) was previously shown to be highly effective in inducing a protective immune response in Japanese flounder (Paralichthys olivaceus) when used as a DNA vaccine. Our previous cDNA microarray analysis demonstrated that interferon-stimulated genes (ISGs) were strongly induced by the HIRRV G protein gene (pHRV-G) but not by the N protein gene (pHRV-N). However, the molecular basis for the difference in protective immunity between pHRV-G- and pHRV-N-vaccinated fish during HIRRV infection is still unclear. In this study, we use a DNA microarray to analyze differences of gene expression in pHRV-G- and pHRV-N-vaccinated fish during HIRRV infection. Microarray analyses showed substantial difference in gene expression patterns during HIRRV infection between fish vaccinated with pHRV-G and pHRV-N. In addition, genes having homology to mammalian T cell activation-related genes were up-regulated in the HIRRV G protein-vaccinated group.     

乙型脑炎病毒SXBJ07株E基因的克隆及原核表达

为构建乙型脑炎病毒(JEV)SXBJ07株E基因的原核表达载体,并在大肠杆菌中进行高效表达;根据JEV SXBJ07株基因组全序列设计1对特异性引物,RT-PCR扩增E基因全长;将目的基因插入pGEM-T连接载体,筛选出阳性重组质粒;将该质粒克隆至原核表达载体pET32α中,再转化入大肠杆菌BL21,经IPTG诱导表达后对其产物进行SDS-PAGE电泳分析和Western-blot检测。结果表明:扩增到了全长为1 500 bp的JEV SXBJ株E蛋白基因;重组质粒pET-32α-E构建成功;融合蛋白可以与乙脑阳性血清抗体特异性结合。说明在大肠杆菌中成功表达了JEV E蛋白。     

日本脑炎病毒SA14-14-2株E基因的克隆及原核表达

根据GenBank公布的日本脑炎(Japanese encephalitis virus,JEV)SA14-14-2减毒株E基因的核苷酸序列,设计并合成一对特异性引物,采用RT-PCR方法扩增其E基因全长cDNA,将扩增产物克隆入pUCm-T载体中,测序后亚克隆到原核表达载体pET-32a( ),筛选重组质粒,转化大肠埃希菌BL21(DE3)宿主菌.经IPTG诱导,SDS-PAGE分析表达产物.结果获得了含全长日本脑炎病毒E基因的重组质粒,经测序证实,与GenBank上E基因序列的同源性达到100%.所表达的融合蛋白主要以包涵体形式存在,为制备JEV实验室诊断抗原打下了基础.     

不同氨基酸包被方法对牙鲆生长及血浆生化指标的影响

本试验比较研究了晶体氨基酸(CAA)和不同壁材包被的CAA对牙鲆(Paralichthys Olivaceus)摄食、生长及血浆生化指标的影响。以大豆浓缩蛋白(SPC)为主要蛋白源,配制了6种等氮等能饲料。其中,饲料SPC作为对照组,未添加氨基酸;饲料SPCF在饲料SPC基础上添加了2.3?A混合物;饲料SPCC、SPCZ、SPCK和SPCT在饲料SPC基础上分别添加了醋酸纤维酯、玉米醇溶蛋白、卡拉胶和棕榈酸甘油酯包被CAA。试验表明:(1)添加CAA和包被CAA均显著提高了牙鲆摄食率(P<0.05),其中以添加CAA效果最好。(2)添加CAA和包被CAA均提高了牙鲆特定生长率(SGR)、饲料效率(FER)和蛋白质效率(PER),其中以SPCT、SPCC组SGR最高;SPCK、SPCT组FER和PER最高。(3)添加CAA和包被CAA均提高了血浆谷草转氨酶(AST)、谷丙转氨酶(ALT)和碱性磷酸酶(AKP)活性,其中以SPCT组AST和ALT活性最高。(4)添加CAA显著提高了血浆尿素氮(UN)和游离氨基酸(FAA)浓度(P<0.05);CAA包被处理后,UN浓度均显著降低(P<0.05);FAA浓度也不同程度地降低,尤其以SPCT组效果最为显著。结果表明以棕榈酸甘油酯包被CAA效果最好。     

猪乙型脑炎乳胶凝集试验与血凝抑制试验方法的比较

用血凝抑制试验 (HI)和乳胶凝集试验 (LAT)两种方法检测乙型脑炎弱毒疫苗免疫猪血清 ,结果均呈阳性反应。用LAT对来自 12个猪场的 94份猪血清进行了乙型脑炎病毒 (JEV)抗体检测 ,并与HI进行了对比 ,两种方法检测结果阳性符合率和总符合率分别为 90 .3% (5 6 /6 2 )和 88.3% (83/94 ) ,两种方法检测结果差异不显著 (p >0 .0 5 )。对来自无乙型脑炎的 12头健康猪血清进行检测 ,两种方法检测结果均为阴性。结果表明 ,用LAT与HI检测乙型脑炎结果符合 ,前者更为简便和实用。     

Gene and haplotype polymorphisms of the Prion gene (PRNP) in Japanese Brown, Japanese native and Holstein cattle

Polymorphisms in the prion protein gene ( PRNP ) are known to be associated with transmissible spongiform encephalopathies in human, sheep and goats. There is tentative association between PRNP promoter polymorphism and bovine spongiform encephalopathy (BSE) susceptibility in cattle. In this study, we genotyped for six bovine PRNP polymorphic sites including a 23-bp indel in the promoter, a 12-bp indel in the intron 1, two nonsynonymous single nucleotide polymorphisms (SNPs), octapeptide repeats in the coding region and a 14-bp indel in the 3'-untranslated region in 178 animals representing Japanese Brown, Kuchinoshima feral, Mishima, Japanese Shorthorn and Holstein. In 64 Japanese Brown cattle, three indel sites were polymorphic. All of the six sites were monomorphic in Kuchinoshima. The 23-bp and 12-bp indel sites were polymorphic in Mishima cattle. The 23-bp and 14-bp indel sites were polymorphic in Japanese Shorthorn cattle. Both SNP sites were monomorphic in all cattle examined in this study. At the 23-bp indel site, the genotype frequencies of Japanese Brown and Holstein breeds were similar to that of BSE affected cattle. We estimated 12 different haplotypes from these genotypic data. A '23-12-K6S14+' haplotype was the major haplotype in all populations, whose frequencies ranged from 0.50 to 1.00.     

瑶山亚种树鼩ISG15蛋白表达及其多克隆抗体制备

【目的】克隆瑶山亚种树鼩干扰素刺激基因15(interferon stimulating gene 15,ISG15)基因,在大肠杆菌中高效表达并纯化、制备多克隆抗体,为其生物学应用及检测方法的建立奠定基础。【方法】提取瑶山亚种树鼩外周淋巴细胞总RNA,经RT-PCR扩增出ISG15基因,亚克隆到真核表达载体构建重组真核表达质粒pcDNA3.1-ISG15,并瞬时转染仓鼠肾细胞(baby hamster syrian kidney, BHK-21);同时亚克隆到原核表达载体pET-28a(+)构建重组表达质粒pET-28a-ISG15,转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达树鼩ISG15蛋白;重组蛋白经镍离子亲和层析法纯化并免疫小鼠,获得鼠抗树鼩ISG15多克隆抗体,利用Western blotting和间接免疫荧光技术(IFA)检测其反应性。【结果】成功克隆瑶山亚种树鼩ISG15基因,并构建了其真核和原核表达载体,真核表达载体在BHK-21细胞中能高效表达;原核表达载体在大肠杆菌中30℃、0.5 mmol/L IPTG诱导6 h获得重组蛋白(分子质量22 ku),...     

珠芽蓼内生固氮菌鉴定及其固氮基因分析

从东祁连山高寒草地采集优势植物珠芽蓼(Polygonum viviparum)并对其内生固氮细菌进行分离、筛选和鉴定。结果表明:从珠芽蓼内分离到14株内生细菌,其中Z22、Z24、Z25、Z27、Z28、Z211、Z214共7株内生细菌具有固氮能力,对菌株Z22的固氮基因(nifH)进行了PCR扩增、测序和系统发育分析,并在GenBank中登录(登录号为EU693342);Z22菌体球形,革兰氏阳性,产芽孢,结合生理生化特征及16SrDNA基因序列测定和同源性分析,鉴定其为球形芽孢杆菌(Bacillus sphaericus),该菌株的16SrDNA序列在GenBank中登录(登录号为EU236749)。本文首次获得了球形芽孢杆菌编码铁蛋白的nifH基因序列。     

Immunohistochemical and ultrastructural characterization of the ovarian surface epithelium of Japanese quail (Coturnix japonica)

Contrary to humans, most ovarian tumors in other species do not arise from the ovarian surface epithelium but are of follicular‐, stromal‐ or germ‐cell origin. One of the few species where ovarian cancer arises from the ovarian surface epithelium (OSE) is chicken (Gallus domesticus). Little is known about the morphology of the OSE in other avian species. In our study we analyzed the OSE morphology of Japanese quail (Coturnix japonica) using ultrastructural and histochemical techniques. Carbohydrate residues have been studied by using a panel of fluorescein isothiocyanate labeled lectins. Japanese quails are commonly used animal models in biomedical research as their housing is comparatively inexpensive and they show a short generation interval. Our ultrastructural and histochemical results demonstrate that the quail ovarian surface epithelium shows characteristic features which resemble the epithelia of both chicken and human. Additionally, the ovarian surface epithelium of the Japanese quail contains cytokeratin as well as vimentin intermediate filaments in their cytoplasm. Therewith and among other parts the quail OSE shows many characteristic features also seen in those of humans, which may qualify quail's ovary as a potential animal model for human ovarian carcinomas.     

Distribution, density and histochemical profiles of the lung mast cells during the post-hatching period of Japanese quails (Coturnix coturnix japonica)

The distribution, density and histochemical characteristics of mast cells in the lungs of the Japanese quail were investigated during the post-hatching period. In the period starting from the first to the 60th day post-hatching, based on proteoglycan content, three types of mast cells, which were alcian blue-positive, safranin O-positive and alcian blue/safranin O-positive, were found to exist in the lungs. The application of staining with berberine sulphate demonstrated that, similar to the distribution of safranin O-positive cells, the heparin-containing cells were located in the periphery of large blood vessels. The percentages of mast cells in different localization sites of the lungs were found to vary with age in the post-hatching period with toluidine blue staining. The lack of any statistically significant increase/decrease in the number of mast cells per unit area of the right and left lung lobes is partially in favour of the proposal that the mast cell number increases with the growth of the lung volume in the post-hatching period.     

Identification of the tet(B) resistance gene in Streptococcus suis

The tetracycline resistance gene, tet(B), has been described previously in Gram negative bacteria. In this study tet(B) was detected in plasmid extracts from 17/111 (15%) Streptococcus suis isolates from diseased pigs, representing the first report of this resistance gene in Gram positive bacteria.     

日本鳗鲡脾脏抗菌肽抗菌活性检测方法的比较

本试验旨在建立一种在日本鳗鲡脾脏抗菌肽分离纯化过程中简便、灵敏的检测其抗菌活性的方法。试验以日本鳗鲡脾脏分子质量小于10 ku的蛋白质为抗菌肽样品,选择琼脂板孔穴扩散法和微孔液体培养法2种传统方法及微量液体培养法为检测方法,比较这3种方法在抗菌肽对3种鳗鲡常见致病菌株(迟钝爱德华菌、气单胞菌、嗜水气单胞菌)抗菌活性检测中的灵敏度和优缺点。结果表明:与琼脂板孔穴扩散法及微孔液体培养法相比,微量液体培养法简便、易于观察结果,具有最高的灵敏度且样品用量最少,适合在鳗鲡脾脏抗菌肽分离纯化过程中跟踪检测抗菌活性,尤其适合分离纯化后期获得微量单一样品抗菌活性的检测。由此得出,微量液体培养法适用于检测日本鳗鲡脾脏抗菌肽的抗菌活性。     

饲料中不同维生素D含量对鲈鱼幼鱼生长性能和钙磷代谢的影响

本研究旨在探讨饲料中不同维生素D含量对鲈鱼幼鱼生长性能和钙磷代谢的影响。以初始体重为(2.26±0.03)g的鲈鱼幼鱼为试验对象,随机分为6组(每组3个重复,每个重复15尾鱼),饲喂维生素D含量实测值分别为34.2、219.4、393.8、775.9、1 534.1和3 091.2 IU/kg的等氮等能饲料,进行9周的养殖试验。结果表明:1)当饲料中维生素D含量在34.2~393.8 IU/kg时,鲈鱼的增重率随着饲料中维生素D含量的升高显著上升(P0.05),但是当饲料中维生素D含量高于393.8 IU/kg时,鲈鱼的增重率变化不显著并且出现平台期(P0.05)。同样,鲈鱼的特定生长率、饲料效率和蛋白质效率均表现出与增重率相似的变化趋势。2)饲料中维生素D含量显著影响了鱼体粗灰分、钙和磷含量,脊椎骨、鳃盖骨和鳞片粗灰分含量以及脊椎骨钙和磷含量(P0.05),但对鱼体粗蛋白质、粗脂肪和水分含量没有显著影响(P0.05)。3)饲料中维生素D含量对鲈鱼血清碱性磷酸酶活性及羟脯氨酸、钙离子和无机磷含量均有显著影响(P0.05)。4)饲料中维生素D含量显著影响了鲈鱼肝体指数及肝脏脂肪和维生素D含量(P0.05)。随着饲料中维生素D含量的升高,肝脏维生素D含量呈现升高的趋势,并在饲料中维生素D含量达到1 534.1 IU/kg时变化趋于平稳。以增重率为评价指标,采用折线模型分析得出鲈鱼幼鱼获得最大生长时对饲料中维生素D的需求量为431.0 IU/kg饲料。以肝脏维生素D含量为评价指标,采用二次曲线模型分析得出鲈鱼幼鱼获得最大肝脏维生素D沉积量时对饲料中维生素D的需求量为2 444.4 IU/kg饲料。     

传染性支气管炎病毒纤突蛋白S1基因的T/A载体克隆策略

参考Genbank收录的IBV纤突蛋白 (S1)基因序列 ,自行设计合成一对引物 ,对传染性支气管炎病毒 (IBV)江苏省地方分离毒株 (JS/95/0 3)RNA进行RT PCR扩增 ,产物经琼脂糖凝胶电泳分析 ,呈现一条 1716bp的条带 ,将其克隆入T/A质粒pMD18 T载体中 ,转化大肠杆菌JM10 9,挑选阳性克隆 ,用质粒少量提取法提取重组质粒 ,用EcoRⅠ和HindⅢ双酶切对重组克隆质粒进行鉴定 ,然后进行序列测定 ,证实为S1基因。将此重组质粒命名为pMDJS950 3S。     

Haplotype C of growth hormone (GH) gene in Japanese Black cattle: Structure of GH protein and a novel method for detection of the gene

From a series of studies on single nucleotide polymorphisms (SNPs) in the bovine growth hormone (GH) gene of Japanese Black cattle, the type‐C (127^Val and 172^Met) that is specific for this breed has been intensively focused upon because of the economic importance for carcass traits, such as intramuscular oleic acid contents. In the present study, we intended to analyze the 3‐D structure of GH of haplotype C, and developed a novel method to detect the type C gene. Three‐D analysis of the type C protein showed that the amino acid residues (127^Val and 172^Met), which are present in the third and fourth helixes, respectively, and are important for binding with GH receptors, are shifted to deeper positions in the molecule compared with that for type A (127^Leu and 172^Thr), implying the alteration of binding interaction with receptors. A novel, efficient and cost‐effective method (Dot‐blot‐SNP technique) for type C genotyping was successfully established, of which the basal method was a reported genotyping of SNPs for a large number of plants, reducing the cost to 10% or less of direct sequencing.