Trypsin and Chymotrypsin are Involved in the Progesterone‐Induced Acrosome Reaction in Canine Spermatozoa

Acrosomal proteases allow the spermatozoon not only to cross the cumulus cells and penetrate the zona pellucida of the oocyte, but also they are needed for the acrosome reaction process (AR). The present study evaluated in vitro the role of trypsin and chymotrypsin in the acrosome reaction of canine spermatozoa by means of protease inhibitors. Spermatozoa obtained from the second fraction of the ejaculate and devoid of seminal plasma were re‐suspended in canine capacitation medium (CCM) and incubated at 38.5°C in 5% CO₂. After 2 h (period of sperm capacitation), aliquots of sperm suspension were incubated separately with trypsin inhibitor NPGB (p‐nitrophenyl‐p′‐guanidino‐benzoate); TI (Trypsin inhibitor I‐S Type from soybean) and with chymotrypsin inhibitor TPCK (N‐tosyl‐L‐phenylalanine‐chloromethyl‐ketone) for 30 min. The AR was induced with progesterone and evaluated using the dual fluorescent staining technique ‘Hoechst and chlortetracycline’. Acrosomal exocytosis levels were statistically significant higher in the samples treated with progesterone than in the control without inducer. However, the trypsin inhibitors NPGB, TI and the chymotrypsin inhibitor TPCK reduced the percentage of AR when compared with the control with progesterone and without inhibitor (p < 0.001), where the AR values were 45.63 ± 3.8%, 51.63 ± 2.8%, 58.38 ±4.1% and 71.25 ± 4.9%, respectively. These results show that trypsin and chymotrypsin inhibitors are effective in blocking the acrosome reaction induced by progesterone in canine; in addition, they suggest the participation of respective proteases in the AR process in this species.     

肽聚糖识别蛋白研究进展

肽聚糖识别蛋白(PGRPs)是一类可识别肽聚糖和含肽聚糖的细菌的模式识别受体,在天然免疫应答中发挥着重要的识别和调节功能.机体通过有限的模式识别受体迅速识别大量不同的病原微生物,进行及时有效的初步防御.近年来,学者们对昆虫和哺乳动物PGRPs已有一定程度的研究,成功地克隆出多个PGRPs基因.对肽聚糖识别蛋白的认识,揭开了天然免疫系统研究的新篇章,对于了解整个免疫系统的反应机制有重要意义.文章就PGRPs基因、结构、表达、功能及进化等方面做了简述.     

重组类丝状蛋白在大肠杆菌中的表达及其溶液态结构分析

利用基因重组技术表达了具有[(GVPGV)2GG(GAGAGS)3AS]n一级结构的类丝状蛋白质(BcEn),该蛋白是把源于弹性蛋白的氨基酸序列GVPGV插入到蚕丝蛋白的结晶序列GAGAGS中,以期利用该弹性片段来调控类丝状蛋白质材料高级结构的形成以及结晶程度,从而达到控制材料性能的目的。利用园二色CD光谱对BcEn在溶液状态下的结构进行了初步分析,结果显示在对BcEn具有良好溶解性的氟有机物水合六氟丙酮中,其结构的形成依赖于BcEn分子量的大小,分子量越大越易形成比较稳定的α-螺旋结构。     

Identification of a Splice Variant of gp100 Expressed in Canine Melanoma Tumours

Introduction:  The melanoma‐associated antigen (MAA) gp100 is a glycoprotein expressed by melanocytes and is considered to be an important target for anti‐tumour immunity in human melanoma. The aim of the current study was to characterise the canine gp100 gene with a view to its inclusion in a DNA vaccine for treatment of canine oral malignant melanoma.
Methods:  RNA was extracted from eight canine oral melanomas and seven control samples (pigmented oral mucocutaneous tissue) and cDNA synthesised. PCR was performed using human gp100 primers designed to generate a 421base pair (bp) fragment. In addition to the expected pcr product, a smaller amplicon (∼260 bp) was present in all tumours and control tissues. Both PCR amplicons were cloned and sequenced. The gp100 genomic DNA sequence was identified from the dog genome resource (NCBI).
Results:  The 421 bp fragment of canine gp100 shared 90.8% identity with human gp100. This partial sequence was located on dog chromosome 10 (CFA10_contig 2982). Further analysis, using the human gp100 gene as a reference, allowed the complete canine gp100 coding sequence to be identified from the dog genome. Sequence analysis of the small PCR product showed it to be a splice variant of gp100 with exon 5 deleted.
Conclusion:  The canine gp100 sequence has been identified and this will allow DNA vaccine constructs containing this MAA to be developed. The splice variant of gp100 with exon 5 deleted was not tumour‐specific and was expressed in both melanomas and normal pigmented tissue.     

母猪妊娠早期血液中差异表达蛋白的鉴定

为了研究母猪妊娠早期外周血液中蛋白质的表达情况,寻找潜在的母猪妊娠早期生物标志物,本试验采集了 8头健康、生产表现正常且体况相近的配种后15 d妊娠与非妊娠的长大二元经产母猪外周血液,分离蛋白,利用iTRAQ技术及生物信息学方法筛选与胚胎附植相关的差异表达蛋白,用ELISA验证其表达情况,并进行ROC曲线分析.结果显示...     

小熊猫犬瘟热病毒感染的诊断

对某动物园发病死亡小熊猫的病料进行电镜检查 ,发现了犬瘟热病毒 ( CDV)样病毒粒子 ;病料用 RT-PCR检测 ,均为 CDV阳性 ,并从部分病料中分离出了 CDV,说明小熊猫存在 CDV感染。用从小熊猫肝脏病料中分离的 CDV LP株接于幼犬 ,结果所有接种犬均发生了犬瘟热。中和试验结果表明 ,LP株是一免疫原性强的 CDV株。     

Expression of ABC-Transport Proteins in Canine Mammary Cancer: Consequences for Chemotherapy

Intrinsic or acquired drug resistance is a major barrier for chemotherapy of cancer. Importantly, the presence of ATP-binding cassette, ABC-transport proteins in tumour cells circumvents an intracellular accumulation of chemotherapeutic drugs. In this study, 103 canine mammary tumour probes were investigated for mRNA expression of seven ABC-transporters by RT-PCR. All tumour samples expressed multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). MRP7 was detected in 97.1% of tumour probes, MRP3 in 96.1%, Pgp in 92.2%, MRP5 in 85.4% and MRP6 in 64.1%. More of the half of tumour samples (56.1%) expressed all of the examined ABC-transport proteins. Approximately one-third of the tumour samples (32.7%) were lacking in one transporter and only 11.2% possessed from three to five transporters. The canine transporter cBCRP was functionally analysed in stable transfected Madin-Darby canine kidney-II cells using an MTT viability test. cBCRP transfected cells showed a 5.4-fold resistance to 10 μ m doxorubicin. Cell survival in the presence of methotrexate was not affected by cBCRP. In conclusion, absence of efficiency of chemotherapy of canine mammary cancer can be caused by expression of seven various ABC-transport proteins. Because cBCRP is expressed in all examined tumour probes and induces resistance to doxorubicin, the application of doxorubicin for treatment of canine mammary is inappropriate.     

不同秋眠型苜蓿顶芽中特异表达蛋白筛选及其功能鉴定

为了获得秋眠时期秋眠型和非秋眠型苜蓿(Medicago sativa L.)顶芽中各自特异表达蛋白,鉴定其在苜蓿生长和秋眠中的功能,同时证明常规高通量筛选差异蛋白不包括特异表达蛋白,本研究拟分析秋眠时期秋眠1级苜蓿标准品种'M averick'和9级苜蓿标准品种'Cuf101'顶芽蛋白质组结果中各蛋白在两品种顶芽中是否...     

Identification of Differentially Expressed Genes and Lipid Metabolism Signaling Pathways between Muscle and Fat Tissues in Broiler Chickens

In this study, signaling pathways and key differentially expressed genes (DEGs) involved in lipid metabolism in muscle and fat tissues were investigated. Muscle and abdominal fat tissues were obtained from 35-day-old female broilers for RNA sequencing. DEGs between muscle and fat tissues were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of DEGs were performed. A total of 6130 DEGs were identified to be significantly enriched in 365 GO terms, most of which were involved in biological processes, cellular components, and molecular functions in muscle and fat tissues. Three important lipid signaling pathways (pyruvate metabolism, the insulin signaling pathway, and the adipocytokine signaling pathway) were identified among the fat and muscle tissues of broilers. The key common DEGs in these pathways included phosphoenolpyruvate carboxykinase 2 (PCK2), acetyl-CoA carboxylase 1 alpha and beta (ACACA and ACACB), and the mitogen-activated protein kinase (AMPK) gene family. Hence, our findings revealed the pathways and key genes and gene families involved in the regulation of fat deposition in the muscle and fat tissues of broilers.     

BmNPV多角体对稳定转化细胞表达荧光蛋白的包埋现象初探

为了探索家蚕核型多角体病毒(BmNPV)多角体对外源蛋白的包埋特性,构建了含有绿色荧光蛋白基因(gfp)和红色荧光蛋白基因(DsRed)的表达载体pIZT-DsRed,用该载体转染家蚕卵巢细胞系(BmN),并以吉欧霉素(zeocin)筛选得到稳定表达绿色荧光蛋白和红色荧光蛋白的BmN细胞系。进一步以野生型BmNPV感染pIZT-DsRed转化BmN细胞,发现部分多角体可以发出绿色荧光和红色荧光,Western blot检测证明多角体中含有GFP蛋白,显示BmNPV多角体可以包埋进病毒粒子以外的其他蛋白成分,表明BmNPV多角体蛋白的包埋机制中存在非特异性识别包埋的现象。     

利用正交试验设计研究葡萄糖处理豆粕对奶牛过瘤胃蛋白的影响

以3头装有永久性瘤胃瘘管的中国荷斯坦牛为试验动物,采用尼龙袋法研究在不同因素下葡萄糖处理豆粕,在奶牛瘤胃内不同时间点DM、CP消失率及其有效降解率的作用效果。结果表明:在不同时间点,提高葡萄糖添加量可使DM、CP消失率逐渐降低,所有试验组与未处理组差异均显著(P0.05),葡萄糖添加量、加热温度、加热时间、含水量四个因素均对蛋白质降解率影响显著,四个因素的影响顺序为:加热温度加热时间葡萄糖添加量含水量;随着加热温度升高、加热时间的延长、葡萄糖添加量的增加,豆粕DM降解率与瘤胃蛋白降解率都逐渐下降。     

Insulinoma and Subclinical Peripheral Neuropathy in Two Dogs

Two dogs with diffuse, subclinical polyneuropathy associated with insulinoma are reported. Seizures were the dominant sign of central nervous system disease. One dog had clinical signs of facial nerve paralysis. Lesions in selected appendicular and cranial nerves included a mixture of demyelination, remyelination, and axonal degeneration. The incidence (range: 18-47%) of these changes far exceeded that of comparable nerves from six control dogs (range 0-11%). Myopathic and electrodiagnostic findings were compatible with the nerve changes.     

2株毒力不同M.bovis差异表达蛋白的定量检测及分析

为研究牛分枝杆菌N和C68004菌株间蛋白水平的差异表达对其致病性的影响,本试验采用串联质谱标记(TMT)定量蛋白组学技术,对2个菌株的毒力及其致病性进行鉴定与分析。结果分析得到2 174个共有蛋白,其中有479个差异表达蛋白(P<0.05)。GO分析结果显示,表达差异显著蛋白的功能包括催化、结合、运输、转录调控以及分子结构相关作用,特别是对内部或外部刺激产生的反应。KEGG分析结果显示,表达差异显著蛋白主要参与代谢和生物过程的调节。导致N菌致病性增强的毒力基因可能是mmaA4、ecce1、IpqY、hspX、Mpb63和mmpl4。并且N菌的耐药基因rpoA、rpoB和rpoC表达量显著高于C68004,可以推断N菌对抗结核一线药物利福平的耐药性可能比C68004更强。随着临床用药频繁和免疫应答的改变,N菌在适应环境过程中,与代谢、DNA复制以及耐药相关的蛋白表达出现了差异。     

葡萄糖处理豆粕对蛋白质保护效果的研究

以3头装有永久性瘤胃瘘管的中国荷斯坦奶牛为试验动物,采用尼龙袋法研究分别添加0、1%、3%、5%、7%、9%葡萄糖保护豆粕,对奶牛瘤胃内不同时间点DM、CP消失率及其动态降解率的作用效果。结果表明:在不同时间点下,提高葡萄糖添加量可使DM、CP消失率逐渐降低,所有试验组与未处理组差异均极显著(P<0.01),DM、CP有效降解率降低最高分别达28.78%和24.82%,不同添加量下,添加量为7%、9%的2组组间差异不显著(P>0.05)。本试验结果证明,葡萄糖加热处理是一种有效保护豆粕蛋白质的方法,且葡萄糖的适宜添加水平为7%。     

Meiotic Response of In vitro Matured Canine Oocytes under Different Proteins and Heterologous Hormone Supplementation

The impact of TCM‐199 supplemented with different proteins and heterologous hormones on the in vitro maturation (IVM) rate of bitch oocytes was evaluated by nuclear staining under fluorescence microscopy. Oocytes were recovered by slicing of ovaries from bitches presented at various stages of oestrous cycle to ovariohysterectomy. The basic culture medium was TCM‐199 supplemented with 25 mM Hepes/l, with 10% heat‐inactivated oestrous cow serum (ECS), 50 μg/ml gentamicin, 2.2 mg/ml sodium bicarbonate and 22‐μg/ml pyruvic acid, 1.0‐μg/ml oestradiol (E 8875; Sigma), 0.5‐μg/ml follicle‐stimulating hormone (FSH) (Folltropin‐V; Vetrepharm Inc., Ontario, Canada) and 0.03 IU/ml human gonadotropin (hCG) (Profasi HP; Serono, Aubonne, Switzerland). Oocytes were distributed randomly between basic culture medium (control) and the corresponding experimental treatment. Hormone treatments were: oocytes cultured in; (1) medium without FSH, (2) control medium supplemented with 20 μg/ml oestradiol, or (3) medium supplemented with 1 μg/ml human somatotropin (hST; Humatrope, Lilly, Saint Cloud, France). The second experiment consisted of oocytes cultured in medium supplemented with 0.4% (w/v) bovine serum albumin (BSA, fraction V; Gibco Grand Island, NY, USA) instead of ECS, or oocytes cultured in medium with 10% inactivated oestrous bitch serum (EBS) instead of ECS. Oocytes were cultured in 100 μl droplets (up to 25 oocytes per drop) under mineral oil at 37°C in a 100% humidified atmosphere containing 5% CO₂ in air. After 72 h of IVM, the highest rates (p < 0.05) of meiotic resumption were achieved with the 0.4% BSA supplementation. A positive influence on the metaphase II (MII) acquisition rate was observed with hST supplement. Oocytes cultured with 10% EBS supplementation did not develop to the MII stage. The results in this study show that the protein and hormone supplements to TCM‐199 culture medium tested did not promote the final steps of IVM of bitch oocytes.     

新城疫病毒人工感染鹅脾脏差异表达蛋白质组初步分析

脾脏是新城疫病毒(Newcastle disease virus,NDV)感染的重要靶器官,本试验旨在从分子水平上分析NDV与宿主之间的相互作用,探寻早期基因Ⅳ型强毒Herts/33和晚期基因Ⅶ型强毒JS5/05造成鹅脾脏病变差异的相关蛋白。30日龄非免疫鹅分别人工感染NDV强毒株Herts/33和JS5/05,并于感染后36、72、108h采集2个感染组和对照组的鹅脾脏,提取脾脏蛋白,以17cm、pH5～8的IPG胶条进行二维电泳,运用PDQuest 8.0.1软件对凝胶图谱进行差异蛋白分析。结果显示:与对照组相比,Herts/33感染组和JS5/05感染组脾脏组织分别有154个和148个蛋白出现了显著的差异表达,其中有86个蛋白点是不同感染组共有的差异点,包括52个感染后上调表达蛋白点,34个下调表达蛋白点;另外,有130个差异蛋白点为NDV感染鹅后不同毒株之间产生的差异表达,包括71个感染后上调表达蛋白点,59个下调表达蛋白点。基因Ⅳ型NDV强毒和基因Ⅶd亚型NDV强毒分别感染鹅后,能引起宿主脾脏组织蛋白表达谱发生不同的改变,这为进一步研究Ⅶd亚型NDV对水禽致病性增强的机制提供了重要线索。     

应用电镜技术和血凝试验检测犬细小病毒和犬腺病毒

电镜技术和血凝试验是检测犬细小病毒和犬腺病毒快速而简便的方法。本试验结果表明,两种方法检验结果大部分相符,但各具特色。电镜技术的优势在于能够直观地显示目标病毒的生长发育、粒子的完整性以及有无非目标病毒、细菌、支原体等污染情况,这对于动物病毒疫苗种毒质量的检测是十分重要的。     

不同羊毛纤维直径细毛羊皮肤组织差异表达蛋白质研究

试验旨在研究不同纤维直径的细毛羊皮肤组织中毛囊差异表达的蛋白质,探讨与羊毛细度相关的蛋白质功能。应用双向凝胶电泳技术建立细毛羊皮肤组织中毛囊差异表达蛋白质图谱;运用ImageMaster 2D Platinum软件检测细毛羊毛囊组织差异表达蛋白质;通过质谱鉴定技术和数据库比对等方法开展细毛羊皮肤组织毛囊特异性蛋白质分类和功能鉴定。结果成功鉴定出94个差异蛋白质,其中,相同年龄超细型细毛羊和细型细毛羊皮肤毛囊组织中有73个差异蛋白质;不同年龄的超细型细毛羊毛囊组织中有21个差异蛋白质。按功能属性划分得到了角蛋白、分子伴侣类蛋白、细胞骨架结构类蛋白、14-3-3蛋白、蛋白酶类、肌球蛋白、血清白蛋白和其他蛋白8类功能蛋白。结果提示,应用生物信息学技术分析这些差异蛋白与羊毛纤维直径的内在联系,为优质细毛羊的培育提供直接有效的基因资源信息,对提高羊毛品质具有重要的意义。