牛病毒性腹泻病毒Erns-E2融合蛋白在果蝇S2细胞中的表达与鉴定

目的:利用果蝇S2细胞表达牛病毒性腹泻病毒(BVDV)Erns-E2融合蛋白,并对其抗体结合能力进行鉴定.方法:用RT-PCR方法扩增BVDV NADL株Erns和E2蛋白的编码基因,利用(G4-S)3柔性15肽基因将扩增的2个基因连接,再与昆虫表达载体pMT/BiP/V5-His连接构建重组表达载体pMT/BiP/V5-His-E(MS)-E2,将后者与筛选质粒pCoBlast共转染果蝇S2细胞后表达Erns-E2融合蛋白.并对表达产物进行鉴定.结果:SDS-PAGE结果表明,融合蛋白相对分子质量为76 800;Westem blotting检测表明,该融合蛋白具有与BVDV抗体良好的结合能力.结论:BVDV的Erns-E2融合蛋白能在果蝇S2细胞中进行表达;经鉴定,表达产物具有良好的抗体结合能力,可用于抗原检测.     

有氧运动降低胰岛素抵抗小鼠海马细胞焦亡相关蛋白及炎症因子的表达

本研究通过观察高脂饮食及有氧运动干预后小鼠海马细胞焦亡及炎症相关蛋白的表达情况,探讨运动改善胰岛素抵抗(insulin resistance, IR)的可能机制。选取6周龄C57BL/6J雄性小鼠38只,随机以正常饮食(n=12)或高脂饮食(n=26)喂养12周后进行葡萄糖和胰岛素耐量实验,根据结果将小鼠分为对照组(n=12)、IR组(n=10)和IR有氧运动组(n=10)。IR有氧运动组进行12周的渐增跑台训练。干预结束后麻醉处死小鼠并剥离海马组织,提取蛋白进行Western blot实验,检测细胞焦亡及炎症相关蛋白的表达情况。结果显示:(1)与对照组相比,IR小鼠海马NFκB,炎症小体相关蛋白Nod样受体蛋白3 (Nod-like receptor protein 3, NLRP3)、斑点样蛋白(apoptosis-associated speck-like protein containing CARD, ASC),细胞焦亡相关蛋白proCaspase-1、gasdermin D (GSDMD)、GSDMD-N及炎症因子IL-1β、IL-18均显著升高,而炎症小体相关蛋白NIMA相关蛋白激酶7 (NIMA-related kinase 7, NEK7)及焦亡相关蛋白Caspase-1有升高趋势,但无显著差异。(2)与IR组相比,渐增跑台训练能够显著降低IR小鼠海马NFκB、NLRP3、NEK7、ASC、pro-Caspase-1、GSDMD、GSDMD-N、IL-1β、IL-18的表达。以上结果提示,12周渐增跑台训练能够显著降低IR小鼠海马焦亡相关蛋白及炎症因子表达,抑制细胞焦亡。     

牛病毒性腹泻病毒结构蛋白功能研究进展

牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)基因组为单股正链RNA,编码4种结构蛋白(C、E^rns、E1和E2)和8种非结构蛋白(N^pro、p7、NS2、NS3、NS4A、NS4B、NS5A和NS5B)。牛感染BVDV后可出现腹泻、流产、繁殖障碍、持续感染等症状,该病死亡率较高,对我国乃至世界养牛业均造成了严重影响。因此,本文就BVDV已确定的4种结构蛋白的功能进行了概括性总结,以期为BVDV的深入研究提供理论依据和参考。     

牛病毒性腹泻病毒单克隆抗体的研究

牛病毒性腹泻——粘膜病是世界性广泛流行的奶牛和肉牛的传染病。其病原为牛病毒性腹泻病毒(BVDV),属于披膜病毒科的瘟病毒属,它的许多生物学特性至今还不很清楚。本试验建立了12株分泌抗BVDV的单克隆抗体(McAb)杂交瘤细胞株,并结合免疫转移电泳法和放射免疫沉淀法,初步研究了BVDV的多肽。     

牛病毒性腹泻病毒（BVDV）对牛免疫缺陷病毒（BIV）的激活作用

通过合胞体分析和反转录酶活力测定,首次证明牛病毒性腹泻病毒能激活牛免疫缺陷病毒的复制与表达,并进一步通过转染实验和凝胶电泳漂移分析证明,当ＢＩＶ ＬＴＲ的ＮＦ－ｋ Ｂ区缺失时,ＢＶＤＶ则不能实现其激活作用,ＢＶＤＶ直接或间接诱导牛ＮＦ－ｋＢ因子作用于ＢＩＶ ＬＴＲ的ＮＦ－ｋＢ区实现其激活作用。     

牛病毒性腹泻病毒Erns蛋白的中华仓鼠卵巢细胞表达及免疫原性分析

本研究利用中华仓鼠卵巢(Chinese hamster ovary,CHO)细胞表达系统制备牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV) E^rns蛋白,并分析其免疫原性。以BVDV-1 NADL标准毒株基因序列为基础,构建BVDV E^rns蛋白重组真核表达质粒pcDNA3.1-BVDV-E^rns,转染悬浮培养的CHO细胞,进行上清分泌表达。SDS-PAGE分析E^rns蛋白的表达和纯化,并用抗His单克隆抗体和BVDV阳性血清进行Western blotting鉴定纯化蛋白;进一步使用纯化的E^rns蛋白免疫新西兰大白兔,通过间接酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)和细胞间接免疫荧光(indirect immunofluorescence,IFA)实验检测血清抗体水平及其免疫反应活性,用病毒中和实验测定免疫兔血清的中和抗体滴度。BCA蛋白定量试剂盒检测纯化的E^rns     

AIM2炎症小体介导细胞焦亡的研究进展

细胞焦亡是一种程序性细胞死亡,参与了多种疾病的发生发展,而炎症反应在细胞焦亡中的作用是目前的研究热点。炎症小体是炎症反应的重要组成部分,其中黑色素瘤缺乏因子2 (absent in melanoma 2,AIM2)炎症小体的激活是诱发由含半胱氨酸的天冬氨酸蛋白酶1 (caspase-1)介导的细胞焦亡的重要因素。靶向AIM2炎症小体激活与细胞焦亡可作为临床相关疾病治疗的有效策略,本文综述了AIM2炎症小体介导的细胞焦亡的研究进展。     

NLRP3炎症体与肠道疾病

Nod样受体蛋白3(Nod-like receptor protein 3,NLRP3)炎症体是机体先天免疫的重要感受器,通过诱导炎症反应和细胞焦亡参与宿主免疫防御,并在维持肠道稳态方面发挥重要作用。近年来,NLRP3炎症体在放射性肠炎、炎症性肠病、结直肠癌和其他肠道疾病中的作用引起临床医生与科研人员的广泛关注。本文就NLRP3炎症体的激活机制和其在肠道疾病中的作用机制进行综述,旨在为临床治疗提供新的靶点与思路。     

炎性小体激活与细胞焦亡的研究进展

细胞焦亡是一种依赖天冬氨酸特异性半胱氨酸蛋白酶1(cysteinyl aspartate specific proteinase 1,caspase-1)/caspase-11的程序性细胞死亡方式。炎性小体的激活在细胞焦亡过程中扮演重要角色。当病原体入侵时,核苷酸结合寡聚化结构域样受体(nucleotide-binding oligomerization domain-like receptor,NLR)和黑色素瘤缺乏因子2(absent in melanoma 2,AIM2)等胞内模式识别受体(pattern recognition receptor,PRR)与相应配体结合,导致炎性小体多蛋白复合物组装和caspase-1/caspase-11激活,进而诱导细胞焦亡发生。深入研究炎性小体激活和细胞焦亡的相关机制,对认识炎症性疾病的发生发展非常重要。本文就炎性小体激活与细胞焦亡的研究进展进行综述。     

牛病毒性腹泻病毒NS3基因的序列分析、表达与抗原性鉴定

本研究应用套式RT-PCR方法扩增出牛病毒性腹泻病毒VEDEVAC株编码NS3蛋白的基因,克隆至表达载体pET-30a(+),并进行测序。对瘟病毒属病毒NS3基因进行氨基酸差异性分析,显示平均P-distance为0.07,系统进化树分析表明VEDEVAC株隶属于BVDV1型。将构建成功的重组质粒转化大肠埃希氏菌Rosetta(DE3),在IPTG诱导下表达NS3重组蛋白,经Ni-NTA亲和层析纯化和尿素梯度透析后进行反应原性鉴定。Western blotting结果显示表达的重组蛋白可以与牛病毒性腹泻病毒阳性血清反应,并与猪瘟阳性血清有交叉反应,ELISA结果显示该重组蛋白具有良好的反应原性。所获得的蛋白为建立针对NS3抗体的ELISA检测方法奠定了基础。     

Autophagy and NLRP3 inflammasome crosstalk in neuroinflammation in aged bovine brains

NLRP3 inflammasome is a multiprotein complex that can sense several stimuli such as autophagy dysregulation and increased reactive oxygen species production stimulating inflammation by priming the maturation of proinflammatory cytokines interleukin-1β and interleukin-18 in their active form. In the aging brain, these cytokines can mediate the innate immunity response priming microglial activation. Here, we describe the results of immunohistochemical and molecular analysis carried out on bovine brains. Our results support the hypothesis that the age-related impairment in cellular housekeeping mechanisms and the increased oxidative stress can trigger the inflammatory danger sensor NLRP3. Moreover, according to the recent scientific literature, we demonstrate the presence of an age-related proinflammatory environment in aged brains consisting in an upregulation of interleukin-1β, an increased microglial activation and increased NLRP3 expression. Finally, we suggest that bovine may potentially be a pivotal animal model for brain aging studies.     

Effect of the bovine viral diarrhea (BVD) virus on the endometrium of the rabbit

The objective of this study was to determine the effect of BVD virus on the rabbit's endometrium. Six New Zealand White does (3-4 kg bwt) were used. Blood samples were obtained before treatment and euthanasia for determination of estrogen and progesterone. Does were anesthetized and both uterine horns identified through a midventral incision. Each horn was doubly-ligated at both cervical and ovarian ends. The right uterine horn (control) was injected with 1ml Eagle's MEM and the left (treated) with 1ml BVD virus (Singer strain, 10(3) CCID(50/ml)). Two does each were euthanized at 48h, 72h and 144h post-inoculation (PI) and uterine samples obtained for viral assay and light microscopic examination. Serum hormonal levels showed that all does were in the estrogenic phase before treatment and euthanasia. Viral isolation was negative for all samples taken. On each day examined, there were no histopathologic lesions in the control uterine horn. However, in the treated horn at 48h PI there was evidence of a purulent endometritis. At 72h and 144h PI there was mononuclear cell infiltration of the stratum compactum, but no other obvious lesions. A common feature in both treated and control uterine horns was mitosis of both endometrial and glandular epithelia. Results of this study suggest that BVD virus can induce histopathologic lesions of the rabbit's endometrium, the most obvious effect being at 48h PI.     

Acid‐sensing ion channels regulate nucleus pulposus cell inflammation and pyroptosis via the NLRP3 inflammasome in intervertebral disc degeneration

ObjectiveLactate accumulation is an important factor in the intervertebral disc degeneration (IVDD). Currently, the effect and underlying mechanism of action of lactate on nucleus pulposus (NP) cell inflammation during IVDD are unclear. Previous studies have found that the NLRP3 inflammasome plays an important role in the regulation of NP inflammation. This study focused on the regulation of acid‐sensitive ion channels (ASICs) in relation to inflammation and the effect of NLRP3 on pyroptosis levels in NP cells under acidic conditions.DesignFor the in vitro experiments, human NP cells were exposed to 6 mM lactate solution; different groups were either treated with NLRP3 inhibitor or transfected with siRNA against NLRP3, siRNA against ASC or a mix of these, and mRNA and protein expression levels were then assessed. For the in vivo experiment, varying concentrations of lactate were injected into rat intervertebral discs and examined via magnetic resonance imaging (MRI) and histological staining.ResultsExtracellular lactate promoted NLRP3 inflammasome activation and degeneration of the NP extracellular matrix; furthermore, it increased the levels of inflammation and pyroptosis in the NP. Lactate‐induced NLRP3 inflammasome activation was blocked by ASIC inhibitors and NLRP3 siRNA.ConclusionsExtracellular lactate regulates levels of intercellular reactive oxygen species (ROS) through ASIC1 and ASIC3. ROS activate the NF‐κB signalling pathway, thus promoting NLRP3 inflammasome activation and IL‐1β release, both of which promote NP degeneration.     

ER stress activates the NLRP3 inflammasome via an UPR-independent pathway

Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1β. This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.     

LncRNA-HOTAIR promotes endothelial cell pyroptosis by regulating the miR-22/NLRP3 axis in hyperuricaemia

Long non-coding RNA (lncRNA) plays an important role in the renal inflammatory response caused by hyperuricaemia. However, the underlying molecular mechanisms through which lncRNA is involved in endothelial injury induced by hyperuricaemia remain unclear. In this study, we investigated the regulatory role of lncRNA-HOTAIR in high concentration of uric acid (HUA)–induced renal injury. We established hyperuricaemia mouse model and an in vitro uric acid (UA)–induced human umbilical vein endothelial cell (HUVEC) injury model. In HUA-treated HUVECs and hyperuricaemia mice, we observed increased HOTAIR and decreased miR-22 expression. The expression of pyroptosis-associated protein (NLRP3, Caspase-1, GSDMD-N, GSDMD-FL) was increased. The release of LDH, IL-1β and IL-18 in cell supernatants and the sera of model mice was also increased. The proliferation of HUVECs stimulated by HUA was significantly inhibited, and the number of TUNEL-positive cells in hyperuricaemia mouse kidney was increased. Bioinformatics analysis and luciferase reporter and RIP assays confirmed that HOTAIR promoted NLRP3 inflammasome activation by competitively binding miR-22. In gain- or loss-of-function experiments, we found that HOTAIR and NLRP3 overexpression or miR-22 knock down activated the NLRP3 inflammasome and promoted pyroptosis in HUA-treated HUVECs, while NLRP3 and HOTAIR knockdown or a miR-22 mimic exerted the opposite effects. Furthermore, in vivo experiments validated that HOTAIR knockdown alleviated renal inflammation in hyperuricaemia mice. In conclusion, we demonstrated that in hyperuricaemia, lncRNA-HOTAIR promotes endothelial cell pyroptosis by competitively binding miR-22 to regulate NLRP3 expression.     

囊泡相关膜蛋白A调控牛病毒性腹泻病毒复制

[背景]牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)是致犊牛腹泻的重要病原之一,而目前BVDV与宿主因子互作机理研究较少,成为限制BVDV防控的重要原因。[目的]探明囊泡相关膜蛋白A (vesicle-associated membrane protein A,VAPA)对BVDV复制的影响。[方法]根据GenBank中VAPA基因,使用Benchling和CHOPCHOP等平台设计靶向VAPA的向导RNA(small guide RNA,sgRNA),融合后克隆至慢病毒lentiCRISPR v2载体中,包装慢病毒后感染牛肾细胞(Madin-Darby bovine kidney,MDBK),使用嘌呤霉素连续筛选5代,使用Western Blot检测VAPA蛋白敲除(knockout,KO)情况;BVDV感染VAPA KO细胞不同时间后,收集细胞提取总RNA,并将等质量的RNA反转录成cDNA,使用实时荧光定量PCR (real-time quantitative PCR,RT-qPCR)和免疫荧光分析(immunofluorescence a...     

鹿源牛病毒性腹泻病毒E0基因的RT—PCR扩增

用RT—PCR方法,成功扩增了鹿源牛病毒性腹泻病毒保护性抗原E0基因。该RT—PCR最佳反应条件分别为退火温度为55℃、25mmol／uL MgCl2（4uL）、10pmol／uL上下游引物（2uL）、5u／uL Taq酶（1uL）。