Hsp90AA1识别狂犬病病毒磷蛋白截短体及磷酸化位点突变体的研究

为了探究热休克蛋白90AA1(Hsp90AA1)识别狂犬病病毒(RV)磷蛋白(P)截短体及磷酸化位点突变体的能力,构建RV P蛋白多种截短体和磷酸化位点突变体,分别与Hsp90AA1真核表达载体共转染于鼠神经瘤母细胞(N2a)。免疫共沉淀分析显示Hsp90AA1能够广泛识别RV P蛋白的多种截短体和不同的磷酸化突变体。结果提示,Hsp90AA1在RABV感染过程中有着广泛的作用,这对解析RABV复杂的复制机理具有重要的意义。     

Pmel17在黑素小体成熟过程中的关键作用

黑色素的形成和沉积主要发生在黑素小体的淀粉样纤维上,前黑素小体蛋白17是黑素小体形成淀粉样纤维的关键蛋白,该蛋白在黑色素的形成和沉积中发挥重要作用。论文对Pmel17在黑素小体成熟过程中的调节作用进行综述,概述了Pmel17的解朊机制,SLC24A5、SLC45A2基因编码的离子交换蛋白对Pmel17解朊的影响,Oa1(ocular albinism type 1)基因对Pmel17表达量的影响,以及部分基因调控Pmel17对黑素小体结构形成的影响。对Pmel17在黑素小体成熟过程中的作用深入研究,有助于探明哺乳动物黑色素的合成和沉积机制,并从Pmel17的角度解析哺乳动物黑色素的色素减退机制和白化现象。     

O型口蹄疫病毒VP1 T细胞与B细胞表位基因双拷贝串联表达产物的免疫应答

以一株O型口蹄疫病毒(FMDV)外壳蛋白VP1基因为模板,合成与细胞免疫及体液免疫相关抗原表位肽基因:21-40肽(20AA)和141-160肽(20AA)基因序列,运用基因工程技术构建了含有串联结构21-40(20AA)～141-160(20AA)～21-40(20AA)～141-160(20AA)的2020-2020VP1融合基因表达载体r2020-2020,转化宿主菌BL21(DE3)RIL后诱导表达,表达产物经SDS-PAGE及Western Blot分析显示重组融合蛋白的分子量约为18Ku.动物实验表明,较小剂量的融合蛋白就能诱导豚鼠产生特异性T淋巴细胞增殖反应及抗FMDV中和抗体,证明该融合蛋白可同时激活细胞免疫及体液免疫反应,具有开发成为抗FMDV疫苗的应用价值.     

补体C9分子原核表达与多克隆抗体的制备

通过大肠杆菌表达系统表达补体C9重组蛋白,并制备其多克隆抗体,用于检测沉积菌体表面的补体C9分子;方法:通过PCR以兔肝细胞cDNA文库为模板扩增获得补体C9部分基因,并将其克隆至原核表达载体pEASY.-Blunt E1,经测序鉴定成功获得重组质粒pBlunt-C9。将其转化至大肠杆菌感受态细胞BL21(DE3)中,经IPTG诱导,Ni-NTA亲和层析柱纯化后,获得重组蛋白。将纯化的重组蛋白免疫BALB/c雌鼠,制备抗C9的鼠源多克隆抗体。结果显示,ELISA测得鼠抗C9多克隆抗体效价为1∶12800,Western Blotting表明,制备的多克隆抗体可以特异性识别沉积在金黄色葡萄球菌和链球菌表面的C9分子,为进一步研究补体的生物学功能以及巩膜复合体(MAC)抗细菌感染机制奠定了基础。     

抗草甘膦转基因豆粕对肉仔鸡肠黏膜免疫的影响

本试验旨在通过研究抗草甘膦转基因豆粕对爱拔益加(AA)肉仔鸡肠黏膜免疫屏障的影响,从黏膜免疫角度评价抗草甘膦转基因豆粕的饲用安全性.选用1日龄AA肉仔鸡360只,随机分成2组,分别饲喂含转基因豆粕和非转基因豆粕的饲粮,试验期21 d.于21 d屠宰取样.采用苏木素-伊红染色法检测肉仔鸡十二指肠、空肠、回肠的小肠绒毛上皮...     

自身抗原表位基因免疫诱导SLE样综合征小鼠模型的建立

通过自身抗原 Sm B/ B′主要表位进行基因免疫 ,诱导系统性红斑狼疮 ( SL E)样综合征的动物模型 ,为进一步探讨 SL E的发病机理奠定基础。以 RT-PCR的方法获得 Sm B/ B′主要表位编码基因 ,构建表达载体 pc DNA3 -Sm B/ B′,基因免疫后以免疫印迹法检测抗 Sm抗体、EL ISA检测抗 ds DNA抗体、免疫荧光法检测肾脏损伤。结果显示基因免疫后小鼠均产生抗 Sm抗体、抗 ds DNA抗体 ,且能诱发动物肾小球 Ig G类免疫复合物的沉积 ,表明自身抗原表位的基因免疫能成功诱导 SL E样综合征小鼠模型。     

抗草甘膦转基因豆粕对肉仔鸡肠黏膜免疫无不良影响

中国农业科学院北京畜牧兽医研究所谭建庄等通过研究抗草甘膦转基因豆粕对爱拔益加(AA)肉仔鸡肠黏膜免疫屏障的影响,从黏膜免疫角度评价抗草甘膦转基因豆粕的饲用安全性。试验选用1日龄AA肉仔鸡360只,随机分成2组,     

积雪草酸通过抑制氧化应激和铁死亡减轻脂多糖诱导的肉鸡心肌损伤的研究

旨在探讨不同浓度积雪草酸(AA)对脂多糖(LPS)诱导肉鸡心肌氧化应激和铁死亡的影响。选取50只1日龄肉鸡适应性饲养至7日龄,并随机分为5组,即对照组(Control)、LPS组(LPS)、低剂量AA组(LPS+AA 15 mg·kg^-1)、中剂量AA组(LPS+AA 30 mg·kg^-1)、高剂量AA组(LPS+AA 60 mg·kg^-1)。所有AA组用相应剂量的AA连续灌胃14 d。在16、18和20日龄时,所有LPS组肉鸡腹腔注射0.5 mg·kg^-1 LPS以建立急性心肌损伤(AMI)模型并于21日龄时处死肉鸡并采集心肌样品。通过苏木精-伊红(HE)染色观察心肌组织病理学变化;使用谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)试剂盒检测心肌组织氧化应激指标;通过实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(Western blot)检测心肌组织氧化应激和铁死亡相关基因和蛋白的表达情况;通过免疫组织化学法检测心肌组织中Nrf2和SLC7A11的阳性表达率;通过免疫荧光法检测心肌组织...     

PEDV中国流行株和疫苗株S蛋白免疫反应性差异分析

为分析PEDV流行株和疫苗株S基因差异对疫苗免疫效果影响,分别在大肠杆菌中表达PEDV流行株CH/ZMDZY/11与疫苗株CV777S蛋白差异较大的N端(22~380aa)区域(SA),将纯化的SA蛋白免疫小鼠获得多克隆抗体,并以SA蛋白为抗原,通过Western Blot及间接ELISA的方法检测2种蛋白与多克隆抗体的反应性差异。结果表明,抗PEDV疫苗株SA蛋白(YSA)多抗与疫苗株SA蛋白的反应性明显高于与流行株SA蛋白(LSA)的反应性;与此相反,抗PEDV流行株SA蛋白多抗与流行株SA蛋白的反应性明显高于与疫苗株SA蛋白的反应性。研究结果提示,PEDV S蛋白22~380aa区域存在差异性抗原表位,PEDV流行株和疫苗株S基因的差异可能是导致基于CV777疫苗株的PED疫苗免疫效果不好的原因。本研究将为开发PEDV流行株亚单位疫苗用于预防临床PEDV流行株的感染提供理论依据。     

水貂脑胞内原虫病肾小球病理形态学研究

用光学、电子显微镜和免疫组织学方法对6只患有脑胞内原虫病的水貂肾脏进行了研究。结果表明,脑胞内原虫感染可引起水貂系膜增生性肾小球肾炎,其发生与免疫复合物颗粒在肾小球基膜和系膜区的沉积有密切关系。     

Pathology of AA amyloidosis in domestic sheep and goats

We describe the main pathologic changes in small ruminants affected by AA amyloidosis, together with the partial sequence of the protein involved. Twenty-one sheep and one goat were selected for presenting macroscopic kidney lesions compatible with systemic amyloidosis. Available tissue samples were studied by histologic, immunopathologic, and ultrastructural means. Renal lesions were characterized grossly by pale cortical surfaces with scattered, miliary, whitish-yellow foci and on cut cortical surfaces by straight, whitish-yellow striations. Gangrenous pneumonia was observed in 16 out of 21 affected sheep (76.2%), although other chronic inflammations were also observed. Amyloid was detected in all grossly affected kidneys using Congo red staining, lesions being most remarkable in glomeruli, affecting 95.5% of animals studied. Congophilic deposits were also observed in intertubular interstitium (68.2%) and medulla (57.1%). All amyloid-affected animals presented proximal convoluted tubule lesions, mostly characterized by an increase in diameter and by hyaline granular degeneration that were responsible for the macroscopic appearance of the kidney. Histologically, amyloid was also seen in blood vessels, spleen, liver, lymph nodes, gastrointestinal tract, and adrenal glands. All amyloid deposits demonstrated greenish-yellow birefringence with polarized light, and the antisera prepared against goat amyloid extracts specifically reacted with birefringent congophilic deposits of both sheep and goats. Ultrastructurally, these deposits were formed by masses of straight, nonbranching fibrils located predominantly in the basement membranes of glomerular capillaries and in the mesangium. Partial sequence of the protein in sheep and goats indicated a high degree of homology with the previously reported sequence of sheep Serum Amyloid A.     

Amyloid angiopathy with cerebral hemorrhage and senile plaque in aged dogs

Amyloid angiopathy with cerebral hemorrhage and senile plaques was found in the brain of aged dogs. In all 9 cases examined, 13 to 19 years old, 6 males and 3 females, amyloid deposits were observed mostly in the wall of cerebral arterioles and capillaries showed hyaline degeneration. The accumulation of amyloid fibrils measuring about 10 nm in diameter was seen in the cerebral vessel wall by electron microscopy. The cerebral hemorrhage was observed in 6 of 9 dogs and 2 of them showed massive hemorrhage. The hemorrhagic foci were sometimes closely contact with the vessels involved in amyloid angiopathy. In addition, senile plaques being classified into 2 types were found in the cerebral cortex of 3 dogs. The first type was characterized by the accumulation of degenerative neurites and often contained granular argyrophilic material. The second type had a well-defined amyloid core with neuritic halo. Amyloid deposits were also found in or around intestinal vessel walls of 3 dogs. The amyloid deposited in the cerebral vessels, senile plaques and intestinal vessels showed characteristic green birefringence under the polarized light even after potassium permanganate treatment.     

Lesions in clinically healthy cattle persistently infected with the virus of bovine viral diarrhea--glomerulonephritis and encephalitis

Four clinically healthy cattle persistently infected with the virus of bovine viral diarrhea were examined for viral antigen and lesions. Antigen was seen by direct immunofluorescence in cytoplasm of the neurons of the brain and cervical part of the spinal cord, cells and basement membrane of renal glomeruli, reticular cells of lymph nodes and spleen, epithelial cells of small intestinal crypts and renal and testicular tubules, and endothelial cells of blood vessels. Infected neurons were pyknotic and surrounded by astrocytes and macrophages. A few blood vessels in the brains were cuffed with mononuclear cells. Basement membranes of renal glomeruli were irregularly thick with eosinophilic material, and mesangial cells in the glomeruli were plentiful. The virus had a direct effect on some tissues, but was restricted in its cytopathogenicity and was not eliminated by defense mechanisms of the host. Renal glomerular lesions were believed to have an immunologic basis.     

Scanning electron microscopy of cellular and acellular glomeruli of male dogs affected with Samoyed hereditary glomerulopathy and a carrier female.

Glomeruli isolated from three male dogs affected with Samoyed hereditary glomerulopathy were compared by scanning electron microscopy with glomeruli of one carrier female and six unaffected dogs. Scanning electron microscopy was performed before and after removal of podocytes and endothelial cells with enzyme and detergent, producing cellular and acellular glomeruli respectively. Cellular glomeruli of unaffected dogs showed podocytes with normally arranged foot processes, while in acellular glomeruli, the subepithelial surface of glomerular capillary basement membranes appeared smooth to finely granular. In contrast, cellular glomeruli of affected males showed microvilli, globular cytoplasmic projections from podocytes, and effacement of foot processes; acellular glomeruli demonstrated ridges and plaque-like irregularities on the subepithelial surface of glomerular capillary basement membranes. Changes in the glomeruli of the carrier female were intermediate between those of unaffected and affected male dogs. The appearance of the subepithelial surface of glomerular capillary basement membranes of acellular glomeruli seen by scanning electron microscopy correlated with the extent of multilaminar splitting of glomerular capillary basement membranes seen by transmission electron microscopy.     

Ultrastructural and enzyme histochemical aspects of amyloidosis in the bovine renal medulla.

Electron micrsocopic studies of bovine kidneys with amyloidosis showed macrophage-like cells in the interstitial amyloid that were either disseminated or in small groups. They had rather uniform protracted vacuoles filled with parallel fibrils. Similar vacuoles were also found in some reticular interstitial cells. In some samples electron-dense vacuoles and vacuoles containing fibrils were seen. Protracted lysosomal enzyme activity was seen in macrophage-like cells and reticular interstitial cells; these areas of activity showed green birefringence with Congo red. The vacuoles were thought to be lysosomes, and the parallel amyloid fibrils could be a consequence of intracellular formation or of phagocytosis.     

不同pH对蛋清溶菌酶形成淀粉样纤维的影响

试验旨在研究不同pH对淀粉样纤维形成的影响。以蛋清溶菌酶（hen egg-white lysozyme,HEWL）为模型蛋白,在不同pH的甘氨酸（50 mmol/L）溶液中（57.0±0.1）℃孵育0~8 d,使其形成不同聚集程度的淀粉样纤维,pH设为2.0、6.5、7.5和8.0;用透射电镜定性观察淀粉样纤维生成状况,采用硫磺素T （ThT）荧光法和刚果红染色法测定淀粉样纤维生长状况,8-苯胺基-1-萘磺酸（ANS）荧光法测定淀粉样纤维疏水性变化,圆二色谱法测定二级结构转化,BeStSel软件预测β-折叠含量。结果显示,透射电镜下观察第8天纤维生长状况发现,pH为2.0时HEWL形成了大量的短杆状淀粉样纤维,同时ThT荧光法结果表明淀粉样纤维生长状况最好（P<0.01）,且生长趋势呈时间依赖性;刚果红染色法显示,在490~510 nm出现红移、540 nm出现特征性肩峰,疏水性显著增强,并且二级结构转化明显,β-折叠含量从天然HEWL蛋白的6.1%增加到37.6%;pH 6.5组仅在透射电镜下观察到极少量的淀粉样纤维,pH 7.5组在视野中未观察到明显的淀粉样纤维,仅观察到大量的球状或杆状蛋白聚集体;pH 8.0组未产生淀粉样纤维。高温条件下,HEWL在强酸性（pH 2.0）条件较易形成淀粉样纤维,且此时疏水性和二级结构转化程度最高,这为解释朊病毒病及其他淀粉样变性疾病的淀粉样纤维形成机制提供了理论基础。     

Localization of extracellular matrix receptors in ICGN mice, a strain of mice with hereditary nephrotic syndrome.

Fibrotic degeneration was examined in the kidneys of ICR-derived glomerulonephritis (ICGN) mice, a novel inbred mouse line with a hereditary nephrotic syndrome of unknown etiology considered to be a good model of human idiopathic nephrotic syndrome. In the present study, we histochemically revealed changes in accumulation of extracellular matrix (ECM) components and in localization of integrins, cellular receptors for ECM, in the kidneys of ICGN mice with the progression of renal failure. Excessive accumulation of basement membrane (laminin and collagen IV) and interstitial (type III collagen) ECM components were demonstrated in the glomeruli and tubulointerstitum of ICGN mice. Marked deposition of type I collagen and tenascin was seen only in the glomeruli of ICGN mice but not in those of ICR mice as normal controls. Increased expression of integrin alpha1-, alpha2-, alpha5- and beta1-subunits in glomeruli with fibrotic degeneration and abnormal distribution of alpha6-subunit were noted in the kidneys of ICGN mice. Excessive laminin, a ligand of alpha6beta1-integrin, was demonstrated on the tubular basement membrane, but alpha6-subunit diffusely disappeared on the basal side of the tubular epithelial cells. We presumed that abnormal integrin expression in renal tubules causes epithelial cell detachment, and consequently tubular nephropathy, and results in disorder of ECM metabolism causing excessive accumulation of ECM components in the kidneys of ICGN mice.     

Diffuse global granulomatous glomerulonephritis in a pig

Diffuse global granulomatous glomerulonephritis with unique morphological characters was detected in a pig. The structure of the basement membrane of glomerular tufts was destroyed in almost all glomeruli. Various inflammatory cells consisted mainly of macrophages infiltrated severely into the glomerular tuft and the Bowman's space of and extended to the periglomerular interstitium. Periarteritis with fibrinoid necrosis was occasionally seen in the arterioles and small arteries running through the renal parenchyma and pelvis. In the present case, the results of either the immunohistochemical reactions to the antigens against PRRSV or PCV-2 or Ziel-Neelsen staining for acid-fast bacilli were negative and no pathogenic bacteria were cultured.     

Immune complex-mediated glomerulopathy in Marek's disease

Chickens infected with Marek's disease (MD) virus developed immune complex (IC)-mediated glomerulopathy. Fluorescent antibody staining technique using antichicken globulin and antichicken complement was used to demonstrate IC in the kidney glomeruli. During the initial stages of MDV infection, IC deposits were seen on the glomerular basement membrane, but subsequently the entire glomerulus was involved. Mesangial cells also had IC deposits. Chicken complement was demonstrated in the glomeruli which had IC deposits. The number of glomeruli with IC deposition was higher in tumor-bearing birds than in non-tumor-bearing birds. Histologically, kidney lesion were characterized by thickening of basement membrane and proliferation of mesangial cells. It is suggested that IC-mediated glomerulopathy might be one of the major causes of death in MD.     

Renal amyloidosis in related English foxhounds

Renal amyloidosis was diagnosed in six related English foxhounds from two separate hunting kennels. There was no apparent age or sex predilection. All the dogs had clinical and laboratory findings compatible with renal disease. The onset of clinical signs was acute in all cases with death or euthanasia in five of the six affected dogs occurring within one week of presentation. The remaining dog was treated with diuresis which produced a temporary improvement. Histological examination of renal tissue from affected dogs identified the presence of both glomerular and interstitial amyloid, which was confirmed by immunohistochemical staining to be of the reactive type (amyloid A). There were occasional sclerosed glomeruli or glomeruli with thickened Bowman's capsules, in addition to interstitial and periglomerular infiltration with a predominance of plasma cells and lymphocytes and secondary changes including tubular eosinophilic casts. Renal papillary necrosis was identified in three dogs. One hound had concurrent multicentric lymphoma. Pedigree analysis of the affected dogs revealed that four individuals shared a common great grandsire and that the same dog was the grand-sire of a further case. Pedigree analysis of five additional dogs from one of the kennels, all of which had clinical signs of acute renal failure but from which renal tissue was not available, revealed a close relationship to those foxhounds with confirmed renal amyloidosis.