干扰素刺激基因15抗病毒免疫研究进展

干扰素刺激基因15(ISG15)蛋白是由干扰素或病原体刺激产生的泛素样蛋白。ISG15能够通过酶促级联反应与靶蛋白共价结合形成ISGylation,称为ISG化。ISG15在干扰素诱导的抗病毒免疫应答过程中发挥重要作用。研究发现,ISG15对多种病毒均具有抗病毒活性。此外,ISG15在细胞自噬、宿主损伤、DNA修复、蛋白翻译等过程中也具有重要作用。论文通过对近年来国内外学者在ISG15类泛素修饰系统、抗病毒免疫分子机制以及ISG15单体生物学功能等方面取得的研究进展进行综述,以期为ISG15抗病毒免疫研究和抗病毒药物的研制提供参考。     

哈兽研发现新的抗猪瘟病毒分子

正2月25日从中国农业科学院哈尔滨兽医研究所获悉,该所研究人员发现了一个可抗猪瘟病毒活性的干扰素刺激基因"鸟苷酸结合蛋白1",并阐明了其抗病毒机制。团队首席科学家仇华吉研究员解释说,该分子发挥抗病毒作用主要依赖其GTPase活性,而猪瘟病毒恰是利用自身NS5A蛋白与干扰素刺激基因相互作用,抑制后者的GTPase活性,进而干扰"鸟苷酸结合蛋白1"的抗病毒功能。专家说,此发现有助于深入了解宿主抗病毒机理,也为揭示猪瘟病毒     

鸡干扰素刺激基因12(ISG12)的克隆表达及抗病毒活性分析

干扰素刺激基因12(interferon-stimulated gene 12,ISG12)在病毒感染过程中已经被监测到,但是针对其抗病毒活性的研究却很少。本研究的主要目的是体外表达鸡的ISG12基因,以禽流感病毒(avian influenza viru,AIV)1215株为病毒模型,利用细胞培养检测其抗病毒活性。利用RT-PCR方法扩增鸡胚成纤维细胞(CEF)的ISG12基因,经测序分析,该基因序列与GenBank中登录的禽属ISG12基因序列相似性为100%。将该基因亚克隆至pET-32a载体构建重组原核表达质粒p32a-C12;工程菌p32a-C12/BL21经IPTG诱导表达,重组蛋白经Ni 2+琼脂糖凝胶柱层析纯化,获得相对分子质量约30 000的目的蛋白。利用ISG12蛋白预处理CEF和鸡外周血淋巴细胞(peripheral blood lymphocyte,PBL)后感染AIV 1215株,或者将该病毒与ISG12蛋白一起接种CEF细胞和PBL细胞,AIV在CEF中的复制能力与对照组无明显差异,而ISG12蛋白预处理鸡PBL细胞和同时感染AIV 1215株时的病毒含量均比对照低。体外抗病毒活性试验表明,ISG12蛋白能够抑制AIV 1215株在PBL细胞中的复制,但在CEF细胞上的作用不明显。     

牛干扰素刺激基因15的生物信息学分析

为了研究牛干扰素刺激基因15编码ISG15蛋白的结构及功能特性,笔者采用生物信息学方法对ISG15基因的开放阅读框进行分析,对ISG15蛋白的氨基酸组成、理化性质、二级结构、三级结构、互作蛋白、亲疏水性、跨膜区、信号肽、亚细胞定位、抗原表位进行分析预测,选取不同动物ISG15蛋白氨基酸序列构建系统进化树。结果表明,该基因全长591bp,开放阅读框全长465 bp,编码154个氨基酸;ISG15蛋白由20中氨基酸构成,分子量为17.31 ku,等电点为6.73,属于酸性蛋白;不稳定系数为50.87,属于不稳定蛋白;二级结构分析显示,ISG15以β-折叠、无规则卷曲为主,均为31.82%;互作蛋白分析显示,ISG15与干扰素、干扰素刺激基因密切相关;ISG15是亲水蛋白,无跨膜结构及信号肽,主要分布于细胞质,细胞核、线粒体、内质网内亦有分布,并预测到7个潜在抗原表位。牛ISG15氨基酸序列与同属偶蹄目的牦牛、瘤牛关系最近,与灵长目类关系最远。     

宿主细胞干扰素刺激基因15蛋白的真核表达及初步应用

为了探究由干扰素刺激基因15(ISG15)编码产生的ISG15蛋白的表达特性,及其对犬瘟热病毒(CDV)感染过程的影响,以及在CDV致宿主免疫抑制过程中的作用,试验设计并合成了针对ISG15基因的特异性引物,提取宿主细胞总RNA,反转录成cDNA后利用RT-PCR方法扩增ISG15全基因序列,扩增产物经琼脂糖凝胶电泳检测、回收纯化目的片段后将其插入到pEGFP-N1真核表达载体中,构建pEGFP-ISG15重组质粒。PCR和双酶切鉴定重组质粒后测序,将阳性重组质粒转染至HEK293T细胞中进行荧光显微镜观察和Western-blot分析。结果表明:经PCR扩增和双酶切鉴定得到与目的片段大小相符的片段,且测序结果证实重组质粒pEGFP-ISG15构建成功,转染HEK293T细胞24 h后可在荧光显微镜下观察到绿色荧光信号,Western-blot可检测到与目的蛋白大小一致的融合蛋白。说明试验构建的重组质粒pEGFP-ISG15可在体外成功表达ISG15蛋白。     

牛源ISG15蛋白的原核表达及抗血清的制备

本研究利用人重组干扰素IFNα-2A刺激牛肾细胞(Mardin-Darby bovine kidney cells,MDBK),并提取细胞总RNA。RT-PCR扩增牛源干扰素刺激基因15(interferon-stimulated gene 15,ISG 15),将其克隆入p Cold-TF表达载体中,转化大肠杆菌BL21(DE3)。经IPTG诱导表达后,获得约70 k Da可溶性ISG15重组蛋白。用试剂盒对重组蛋白ISG15进行纯化回收,经BCA法测定其蛋白浓度为1 mg/m L。用纯化的ISG15重组蛋白免疫ICR小鼠,制备多克隆抗体血清,用间接ELISA方法测定多抗血清效价为1:102 400。利用Western blot进一步鉴定ISG15多抗血清,结果发现其与纯化的ISG15重组蛋白能发生特异性反应,可用于后续研究。     

牛干扰素研究进展

BoIFN是机体在接触病毒或其他诱生物后分泌的一类蛋白，具有广谱抗病毒、抑制细胞增殖和免疫调节等多种生物学活性。BoIFN生物学活性的发挥受其基因的调节和控制，涉及到RNA和蛋白质的合成Ⅲ。干扰素是一个大家族，可以分为许多种类。     

猪繁殖与呼吸综合征病毒通过阻碍STAT1/STAT2核转位抑制Ⅰ型干扰素的信号转导

Ⅰ型干扰素(IFNs),IFN-α/β诱导抗病毒应答,在天然免疫抗病毒感染中起着重要作用。猪繁殖与呼吸综合征病毒(PRRSV)抑制Ⅰ型干扰素的合成,但其是否抑制IFN的信号转导尚未明确。本研究结果发现PRRSV干扰IFN的信号转导通路。用PRRSV VR2385株感染MARC-145细胞,经IFN-α处理的感染细胞中,干扰素刺激基因ISG15和ISG56的转录、信号转导蛋白及转录激活因子(STAT)2水平均显著低于未经过IFN-α处理过的对照感染细胞。在PRRSV感染的细胞中,IFN诱导的STAT1和STAT2磷酸化及其异源二聚体的形成都未受影响,但大部分的STAT1/STAT2/IRF9的三聚体仍存在于PRRSV感染细胞的细胞质中,表明三聚体的核易位受到了阻碍。PRRSV VR2385感染的细胞中过度表达的NSP1β抑制了ISG15和ISG56的表达,并阻止STAT1的核转位,这些都表明NSP1β可能是抑制IFN信号转导的病毒蛋白。PRRSV感染的猪肺泡巨噬细胞(PAMs)也抑制INF-α刺激的ISG基因和STAT2蛋白的表达。相反,已获批上市的的低致病性疫苗株Ingelvac PRRSV MLA感染PAMs,不需要添加外源的IFN就能活化包括趋化因子和抗病毒细胞因子等IFN诱导基因的表达,且检测不出其对IFN的信号转导产生影响。这些发现都说明PRRSV通过阻碍ISGF3的核转位阻止Ⅰ型干扰素的活化和信号转导通路。     

哈兽研研究所:发现猪瘟病毒干扰素

正近日,中国农业科学院哈尔滨兽医研究所研究人员发现了一个可抗猪瘟病毒活性的干扰素刺激基因"鸟苷酸结合蛋白1",并阐明了其抗病毒机制。专家说,此发现有助于深入了解宿主抗病毒机理,也为揭示猪瘟病毒逃避宿主抗病毒策略提供了线索。结合抗病毒干扰素刺激基因开展研究,对创新猪瘟防控策略具有重要意义,并为猪瘟病毒所在黄病毒科其他成员的抗病毒研究提供了参考。此研究结果已在国际著名病毒学杂志     

山羊干扰素刺激基因15克隆、生物信息学分析及亚细胞定位

【目的】对山羊干扰素刺激基因15(interferon-stimulated gene 15,ISG15)的CDS区进行克隆、表达和生物信息学分析,并探讨小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)感染山羊子宫内膜上皮细胞(caprine endometrial epithelial cells,EEC)对ISG15的影响。【方法】根据GenBank中公布的山羊ISG15基因预测序列(登录号:XM_005690795)设计特异性引物,利用RT-PCR方法扩增山羊ISG15基因CDS区,连接至真核表达载体进行测序并表达,对不同物种ISG15核苷酸及氨基酸序列进行比对,并构建系统进化树,利用生物信息学方法对ISG15蛋白理化性质、跨膜结构、修饰位点、二级结构、三级结构、亚细胞定位等进行分析。通过构建真核表达载体转染及PPRV感染EEC细胞,利用间接免疫荧光的方法观察其外源性和内源性亚细胞定位,并探究PPRV感染对EEC细胞的影响。【结果】成功克隆出山羊ISG15基因CDS区并进行了真核表达。山羊ISG15核苷酸和氨基酸相似性及进化树分析都与盘羊和绵羊亲缘关系最近。生物信息学分析结果显示,山羊ISG15基因位于16号染色体上,全长474 bp,编码157个氨基酸,分子质量约为17.47 ku,为亲水性蛋白,无跨膜区和信号肽,有1个潜在的N-糖基化位点,16个潜在的O-糖基化位点和10个潜在的磷酸化位点。二级结构与三级结构预测显示,山羊ISG15蛋白由无规则卷曲、延伸链、α-螺旋和β-转角组成,比例分别为34.39%、31.21%、21.66%和12.74%。可以与干扰素和泛素化相关的蛋白相互作用,并参与机体的抗感染作用。亚细胞定位显示,外源性和内源性ISG15均定位于细胞质中。【结论】试验成功克隆出山羊ISG15基因CDS区序列,亚细胞定位发现内源性和外源性ISG15均定位于EEC细胞的细胞质中。结果为后续ISG15基因细胞内功能研究奠定基础。     

Phase I evaluation of CCNU (lomustine) in tumor-bearing cats

1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor-bearing cats. Response data were obtained when available. Twenty-five cats were treated with CCNU at a dosage of 50-60 mg/m3 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose-limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/microL (mean, 2,433 cells/microL; range, 0-9,694 cells/microL). The time of neutrophil nadir was variable, occurring 7-28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6-week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14-21 days after treatment. The median platelet count at the nadir was 43,500 cells/microL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50-60 mg/m3 every 6 weeks.     

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects

Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.     

Prevalence of gastric lesions in racing Alaskan sled dogs

Human and equine athletes are reported to have a high prevalence of gastric disease, and anecdotal evidence suggests a similar phenomenon applies to racing sled dogs. To investigate the prevalence of gastric disease in racing sled dogs, we conducted 2 gastroscopy studies on dogs competing in the annual Iditarod Sled Dog Race. A pilot study of dogs that were either dropped from the 2000 Iditarod Sled Dog Race because of illness or that finished the race indicated that, approximately 5 days after competing, 10 of 28 dogs (35%) had endoscopic evidence of gastric ulceration, erosion, or hemorrhage. The next year, an endoscopic study of 73 dogs participating in the 2001 Iditarod race was performed in order to evaluate a larger population of dogs. Data from 70 of these dogs could be used; 34 (48.5%) had ulceration, erosion, gastric hemorrhage, or some combination of these findings. When this group of 70 dogs was compared retrospectively to a control group of 87 dogs presented to the Texas A&M University (TAMU) Veterinary Medical Teaching Hospital, the Iditarod sled dogs had a significantly higher prevalence (P = .049) of gastric lesions. These findings suggest that, similar to athletes of other species, elite canine athletes have an increased prevalence of gastric disease compared to the canine population at large.     

The Epidemiology of Gastrointestinal Nematodes of Dairy Cattle in Central Kenya

The epidemiology of H. placei and of other gastrointestinal nematodes in yearling dairy cattle was examined on two farms in Kiambu District, central Kenya during each of 13 one-month periods from April 1993 to April 1994. On each farm, 32 newly weaned dairy calves were given a single dose of albendazole and then placed on experimental pastures. Twelve of the animals were designated for bi-monthly slaughter (n = 2) and analysis of worm population characteristics and 20 were designated for blood and faecal collection and for weighing. Two parasite-free tracer calves were grazed alongside the weaner calves each month throughout the study period and were also slaughtered for analysis of worm populations. Faecal egg counts, haematological and serum pepsinogen determinations, herbage larval counts, and animal live weight changes were recorded monthly. The study revealed that Haemonchus placei, Trichostrongylus axei, Cooperia spp. and Oesophagostomum radiatum were responsible for parasitic gastroenteritis and that H. placei was the predominant nematode present in the young cattle on both farms. Faecal egg counts from resident cattle and necropsy worm counts revealed that pasture larval levels were directly related to the amount of rainfall. The total worm burdens in the animals were highest during the rainy season (March–June and October–December) and lowest during the dry seasons (July–September and January–February). The very low recovery of immature larvae of H. placei from the tracer calves indicated that arrested development is not a feature of the life cycle of this parasite in central Kenya. The maintenance of the parasite population depended on continuous cycling of infection between the host and the pasture. The agroclimatic conditions of the study area were such that, in general, favourable weather conditions for the development and survival of the free-living stages of gastrointestinal nematodes existed all year round.     

Diversity of Ectoparasites in Sheep Flocks in Sa~o Paulo,Brazil

The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of Sa~o Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the `Associaça~o Paulista de Criadores de Ovinos' (ASPACO; Sa~o Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.     

Evaluation of biohydrogenation rate of canola vs. soya bean seeds as unsaturated fatty acids sources for ruminants in situ

An experiment was conducted to study disappearance of C14 to C18 fatty acids, lag times and biohydrogenation (BH) rates of C18 fatty acids of ground soya bean and canola seeds in situ. Three ruminally fistulated Dallagh sheep were used to determine ruminal BH of unsaturated fatty acids (UFAs). Differences in the disappearance of fatty acids through the bags and lag times were observed between the oilseeds. We saw that the longer the incubation time of the oilseeds in the rumen, the lower the content of C18:2 and C18:3. Significantly higher lag times for both C18:2 and C18:3 were observed in ground canola compared to ground soya bean. BH rates of C18:2 and C18:3 fatty acids in soya bean were three times higher than those of canola. These results suggest that the fatty acid profile of fat source can affect the BH of UFAs by rumen micro‐organisms. So that UFAs of canola had higher ability to escape from ruminal BH. It seems that fatty acid profile of ruminant products is more affected by canola seed compared to soya bean seed.     

Serum biochemical values and mineral element contents of tissues in yaks

Serum biochemical values and the wet weight to dry weight ratios of tissues were determined in yaks in Shandan county of Gansu province. The liver, kidney, heart and muscle contents of seven elements in yaks were also analysed. Most serum biochemical values were similar to those of cattle, camels and sheep, but the calcium concentration was considerably above the normal range for other ruminants. The liver contained the highest concentrations of copper, manganese and cobalt and the kidney of selenium, iron and calcium.Abbreviations Alb albumin - ALT alanine aminotransferase - AST aspartate aminotransferase - BUN blood urea nitrogen - Chol cholesterol - Glob globulin - -GT -glutamyl transpeptidase - IP inorganic phosphorus - TP total protein     

Avian brood parasitism — a growing research area in behavioral ecology (part Ⅱ)

正We are pleased to publish the second special issue on avian brood parasitism and to be responsible guest editors for the two special issues of Chinese Birds (Vol. 3, No. 4, 2012 and Vol. 4, No. 1, 2013), entitled "Avian Brood Parasitism - A Growing Research Area in Behavioral Ecology". The first issue was published in December 2012. The goal of the two special issues is to publish accumulated knowledge and some of the recent developments in     

Internal Parasites and Haematological Values in Cattle Slaughtered in Buea Subdivision of Cameroon

Tropical Animal Health and Production -