鹿源BVDV基因E_0蛋白特性分析

以DNAStar5.01软件,对鹿源BVDV基因E0蛋白特性进行了分析。结果表明E0蛋白由227个残基构成,等电点为7.61。E0蛋白中有与地衣类植物核苷酸酶的保守区域相似序列,维持催化活性所必需的氨基酸残基区域在28-40和71-89处,催化活性的氨基酸为His30、His79,且该序列高度保守。E0蛋白氨基酸亲水性与抗原性指数成平行相关,BVDV各毒株间非常相似,亲水性与抗原性指数都较高的区域有8-16,23-29,59-67,70-81,96-109,114-122,127-134,137-143,165-172,186-198和213-221,适合研制亚单位基因工程疫苗。     

牦牛病毒性腹泻病毒E2基因的克隆鉴定及原核表达

运用聚合酶链式反应,以牦牛BVDV基因组DNA为模板扩增出牦牛BVDV E2基因.为研究E2蛋白的抗原性,将E2基因插入到pET-32a原核表达载体,构建重组表达质粒pET-32a-E2,并转化至BL21(DE3)宿主菌中,利用IPTG诱导表达.经SDS-PAGE检测,pET-32a-E2在宿主菌BL21(DE3)中表...     

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E2蛋白单克隆抗体的制备及初步鉴定

为制备牛病毒性腹泻病毒(BVDV)糖蛋白E2单克隆抗体(MAb),利用原核表达并且纯化的重组糖蛋白E2(rE2)免疫BALB/c小鼠,取免疫后小鼠脾细胞与骨髓瘤细胞SP2/0融合.采用以BVDV为检测抗原的间接ELISA筛选阳性细胞克隆,经3次克隆纯化后获得2株稳定分泌抗E2特异性MAb的杂交瘤细胞株,分别命名为4E3与1G11.用4E3与1G11杂交瘤细胞株接种BALB/c小鼠制备腹水,采用rE2及BVDV包被的ELISA测得的效价分别是6.21×106和6.83×105及6.83×105和7.5×104.间接ELISA、Western blot、IFA试验表明两株杂交瘤细胞所分泌的MAb具有良好的反应性和特异性.经抗体亚类鉴定4E3与1G11均为IgM/K.特异性试验表明4E3与1G11这2株MAb均不与牛传染性鼻气管炎病毒、牛副流感病毒3型、牛腺病毒3型反应;其中4E3不与猪瘟病毒反应,而1G11则可与猪瘟病毒发生交叉反应,这种反应特性可试用于BVDV与猪瘟病毒的鉴别诊断.所制备的4E3与1G11 MAb可以用于BVDV抗原的检测,为建立检测BVDV E2蛋白血清抗体的ELISA奠定了基础.     

牛病毒性腹泻病毒的成熟和释放

试验中用电镜观察了牛病毒性腹泻病毒ＯｒｅｇｏｎＣ２４Ｖ株在感染新生牛睾丸细胞中的形态发生。成熟的病毒颗粒是直径约为５０ｎｍ的球形颗粒,内含直径约为３０ｎｍ的核心。病毒在宿主细胞的胞质内复制,通过糙面内质网膜出芽成熟。病毒可以通过外排或在细胞死亡后含有病毒颗粒的空泡崩溃而释放到胞外。     

梅花鹿源BVDV基因E0克隆与序列分析

对梅花鹿源BVDV基因E0进行了克隆和序列分析。结果表明,梅花鹿源BVDV基因E0的大小为681bp,与报道9株BVDV（VEDEVAC、Bega、C24V、ILLC、NADL、OSLOSS、R1935、SD-1、Y546）和7株猪瘟病毒（ALD、Bres-cia、c、GPE、JL、LN9912、SM）及3株羊边界病毒（BD31、C413、BDVX818）相比,核苷酸序列的同源性依次为98.6%～84.8%、76.1%～74.7%、77.0%～76.7%。梅花鹿源BVDV为Ιb基因亚型。     

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。     

牛病毒性腹泻病毒的分子生物学研究进展

牛病毒性腹泻病毒（Bovineviraldiarrheavirus,BVDV）,也称牛病毒性腹泻一粘膜病病毒（Bovinevlraldi。h。一mucosaldi。。virus,BVD一MDV）,在分类学上属黄病毒科（Flaviviri－d。）,瘟病毒属（Pestiv＿）［l］。鉴于其基因结构及分子生物学特征近来也有人提议新设一瘟病毒科［’,’l。BVDV是瘟病毒属的代表病毒,与属内的猪瘟病毒（HOgCh。le。Vi。,HCV;或称CI。icalswinefevervi。s,CSFV）及羊边界病病毒（Borderdi。asevirus,BDV）,在血清学上有交叉反应［‘］,BVDV感染牛可引起多种临床症状,如：高热、流…     

牛病毒性腹泻病毒基因组cDNA文库的构建

以牛病毒性腹泻病毒（ＢＶＤＶ）┥ｎＬ株的基因组ＲＮＡ为模板,经逆向转录酶作用,合成第一链ｃＤＮＡ,再以ＲＮａｄｓｅ／Ｈ与ＤＮＡ聚合酶Ｉ联合作用合成ｄｓｃＤＮＡ,并以ｄＣ同聚物尾化。ｐＵＣ８ＤＮＡ在Ｐｓｔ Ｉ酶解后,以ｄＧ同聚物尾化,两者退火构成重组质粒,转化到Ｅ．ｃｏｌｉ ＪＭ１０１受体菌中,另以γ－＾３２Ｐ－ＡＴＰ标记ＢＶＤＶ ＲＮＡ制备探针,通过菌落原位杂交筛选重组子。酶切分配表明重组质粒插入     

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。     

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.     

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。     

牛病毒性腹泻病毒Erns蛋白激活NOD样受体热蛋白结构域相关蛋白3炎症小体诱发细胞焦亡的研究

【目的】牛病毒性腹泻病毒(bovine viral diarrhea virus, BVDV)是引起牛病毒性腹泻-黏膜病的关键病毒。BVDV的结构蛋白Erns可在病毒感染的初期削弱宿主的免疫防御,引发牛群炎症反应。核苷酸寡聚化结构域样受体(nucleotide-binding oligomerization domain, NOD)热蛋白结构域相关蛋白3 (NLRP3)炎症小体是NOD样受体(NOD-like receptor, NLRs)家族重要成员,调控炎症性疾病的发生发展,同时激活的NLRP3炎症小体能够引起宿主细胞焦亡,进而诱发级联放大的炎症反应。但BVDV Erns蛋白在BVDV感染诱发炎症反应的分子机制尚不清楚。【方法】为进一步探索Erns蛋白对BVDV感染激活NLRP3炎症小体诱发细胞焦亡的影响,构建了BVDV Erns蛋白的真核表达质粒pCMV-HA-Erns,过表达BVDV Erns蛋白,检测BVDV感染细胞中NLRP3炎症小体组分[半胱氨酸蛋白酶(caspase-1)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein, ASC)和NLRP3]、IL-1β的mRNA转录水平和蛋白表达水平,以及细胞死亡调节蛋白(gasdermin D, GSDMD)的基因表达和蛋白剪切情况,并通过扫描电镜观察牛睾丸(bovine testis, BT)细胞膜成孔及BT细胞内容物释放情况,以分析Erns蛋白诱导BT细胞产生细胞焦亡。【结果】Erns蛋白能够显著引起NLRP3炎症小体活化进而激活caspase-1,活化的caspase-1一方面切割GSDMD,形成有活性的GSDMD-N端并在BT细胞膜形成孔洞,释放内容物,诱导BT细胞发生细胞焦亡;另一方面活化的caspase-1切割pro-IL-1β,形成有活性的IL-1β,并释放到BT细胞外,引起BT细胞上清中IL-1β水平上升。【结论】系统解析了BVDV Erns蛋白激活NLRP3炎症小体介导细胞焦亡的产生,对疫苗及治疗药物的研制具有重要指导意义。     

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...     

牛病毒性腹泻病毒1型病毒样颗粒的制备及其对豚鼠的免疫原性分析

为了获得预防牛病毒性腹泻病毒1型(bovine viral diarrhea virus 1,BVDV-1)感染的病毒样颗粒,扩增C-Ems-E1-E2编码区段并克隆至pFastBacDaul载体,转化大肠杆菌DH10Bac感受态细胞与Bacmid重组获得Bacmid-BVDV-1,转染至Sf9细胞,获得重组杆状病毒B...     

Immune response to vaccination with DNA encoding the bovine viral diarrhea virus major glycoprotein gp53 (E2)

Bovine viral diarrhea virus (BVDV) is a worldwide pathogen in cattle which has not been controlled by classical vaccination. The region encoding the BVDV major glycoprotein gp53 (E2) known to possess virus-neutralizing activity was cloned into a mammalian expression vector under the human cytomegalovirus (CMV) intermediate early promoter. Intramuscular and intradermal administration of the recombinant plasmid DNA into BALB/c mice induced BVDV gp53-specific antibody responses to both biotypes (cytopathic and noncytopathic) of BVDV genotype 1, and to cytopathic BVDV genotype 2. BVDV-neutralizing antibodies were generated against BVDV genotype 1 strains and they also persisted 6 months after the last injection.     

Preparation of chicken IgY against recombinant E2 protein of bovine viral diarrhea virus (BVDV) and development of ELISA and ICA for BVDV detection

Bovine viral diarrhea virus (BVDV) infects cattle and may lead to persistent infection (PI). The PI animals harbor BVDV throughout their life and become immune tolerant against BVDV. Thus, diagnosis of this virus in herd is highly important. Recombinant E2 protein expression (using pET-32a in Escherichia coli) was confirmed by SDS-PAGE and Western blotting; then purified by Ni⁺ affinity chromatography. Chickens were immunized with BVDV-E2 protein, and IgY antibodies were extracted from egg yolk by PEG-6000. The peak titer of anti-BVDV-E2-IgY was 1:128,000 after the fifth immunization. IgY-based enzyme-linked immuno sorbent assay (ELISA) and immunochromatographic assay (ICA) were further developed. Coincidence of ELISA and ICA test with RT-PCR was 95.45 and 90.91%, respectively. The anti-BVDV-E2 IgY could be used in routine screening of BVDV infection. Besides, it can also be applicable while licensing and/or using live vaccines; screening of imported products containing bovine serum and strong surveillance of BVDV outbreaks.