Giardiasis is a common waterborne zoonotic disease caused by Giardia intestinalis. Upon infection, Giardia releases excretory and secretory products (ESPs) including secreted proteins (SPs) and extracellular vesicles (EVs). Although the interplay between ESPs and intestinal epithelial cells (IECs) has been previously described, the functions of EVs in these interactions and their differences from those of SPs require further exploration. In the present study, EVs and EV-depleted SPs were isolated from Giardia ESPs. Proteomic analyses of isolated SPs and EVs showed 146 and 91 proteins, respectively. Certain unique and enriched proteins have been identified in SPs and EVs. Transcriptome analysis of Caco-2 cells exposed to EVs showed 96 differentially expressed genes (DEGs), with 56 upregulated and 40 downregulated genes. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) indicated that Caco-2 genes related to metabolic processes, the HIF-1 signaling pathway, and the cAMP signaling pathway were affected. This study provides new insights into host-parasite interactions, highlighting the potential significance of EVs on IECs during infections.

[目的]以羊口疮病毒129(ORFV129)蛋白为研究对象,在构建山羊鼻甲骨原代细胞cDNA文库的基础上,通过酵母双杂交筛选与其相互作用的蛋白.[方法]采用Smart?技术构建山羊鼻甲骨原代细胞cDNA文库.构建诱饵质粒pGBKT7-129,并将其转化至Y2HGold酵母感受态细胞,验证质粒pGBKT7-129是否具有自激活性.对转化了质粒pGBKT7-129的菌液进行生长曲线测定,验证该质粒是否对酵母细胞有毒性作用.以ORFV129为诱饵蛋白,利用酵母双杂交系统筛选出与ORFV129相互作用的宿主胞内蛋白并对阳性菌落进行PCR和测序鉴定.利用DAVID 6.7的GO数据库对胞内宿主蛋白进行功能分类和通路分析,并依据Cytoscape v 3.8.0软件绘制ORFV129与胞内蛋白相互作用的网络图.以山羊脾脏为组织样本,采用RT-PCR技术克隆山羊补体C1q结合蛋白(complement C1q binding protein,C1QBP)基因CDS区序列,并采用在线软件进行生物信息学分析.将C1QBP基因CDS区序列连接至pcDNA3.1(+)构建真核表达载体pcDNA3.1(+)-C1QBP,并将其转染至山羊鼻甲骨原代细胞进行亚细胞定位分析.[结果]试验成功构建山羊鼻甲骨原代细胞cDNA文库,文库容量约为6.0×106 CFU/mL.成功构建诱饵质粒pGBKT7-129,重组质粒pGBKT7-129无自激活能力且对酵母细胞无毒性.利用酵母双杂交筛选出14个与ORFV129进行互作的胞内蛋白并对阳性克隆进行PCR、测序验证.试验成功克隆山羊C1QBP基因CDS区,长度为837 bp,编码279个氨基酸.系统进化树显示,山羊和绵羊亲缘关系最近.C1QBP蛋白为不稳定的亲水性蛋白,无信号肽结构和跨膜结构域,主要包括3种磷酸化位点,分别是18个丝氨酸、4个苏氨酸和3个酪氨酸位点.C1QBP蛋白二级结构由α-螺旋(33.81％)、无规则卷曲(46.40％)、延伸链(16.91％)和β-转角(2.88％)组成,三级结构与二级结构一致.间接免疫荧光试验表明,C1QBP蛋白散在分布于细胞质中.[结论]筛选出了与ORFV129蛋白相互作用且在天然免疫应答中发挥作用的宿主胞内蛋白C1QBP,通过间接免疫荧光试验验证C1QBP定位于细胞质中,推测ORFV129与能C1QBP相互作用诱导炎症,为进一步验证ORFV129蛋白介导ORFV抑制机体免疫应答的过程奠定基础.

The aim of the present study was to clone goat BIRC5, and to reveal the role of BIRC5 gene in regulating the cycle and apoptosis of goat testis cells through the overexpression or interference of BIRC5. These data will be beneficial for exploring the role of BIRC5 gene in goat. The BIRC5 gene sequence was cloned by RT-PCR from spleen tissue of 3 three-day-old healthy Jianzhou male goats with 3 kg of body weight, and sequenced for further bioinformatics analysis using online softwares. The clone recovery product was ligated to a eukaryotic expression vector to construct pcDNA3.1（+）-BIRC5. Effective siRNA was designed and screened based on goat BIRC5 gene sequence. After pcDNA3.1（+）-BIRC5 and siRNA were respectively transfected into goat testicular cells, the expression of BIRC5 protein was detected by Western blot, and cell cycle and apoptosis were detected by flow cytometry. Meanwhile, the expression of apoptosis-related genes Bax, Caspase3, Caspase7, Bcl-2, p53, BCL2L11 and PARP1 were detected by RT-qPCR. A length of 506 bp BIRC5 gene sequence was cloned successfully, including 28 bp of 5′UTR, 429 bp of CDS region, and 49 bp of 3′UTR,encoding 142 amino acids. The phylogenetic tree showed that BIRC5 had the closest relative to Bos taurus. The overexpression of BIRC5 gene inhibited the apoptosis of cells, and the cells were arrested in G2/M+S phase; it also down-regulated the expression of Caspase7, p53, BCL2L11, Bcl-2 and Bax gene mRNA, but did not significantly change the mRNA expression of Caspase3 and PARP1. siRNA interference of BIRC5 gene promoted the apoptosis of cells, and the cells were arrested in G0/G1 phase; it also up-regulated the expression of Caspase3, Caspase7 and PARP1 gene mRNA, and down-regulated the expression of p53 and Bcl-2 gene mRNA, but did not significantly change the mRNA expression of Bax and BCL2L11. Goat BIRC5 gene could inhibit the apoptosis of testis cells and promote the arrest of goat testicular cells in G2/M+S phase. These data may lay a foundation for further study of BIRC5 gene function.

目的:尝试设计制造一种模拟卒中患者典型痉挛的教学仿真机器人原型机,并对其仿真效果进行初步评估.方法:根据简化公式:F=Bθ(t)+Kθ(t)+A设计并制造了教学仿真机器人的原型机.公式中,肌肉张力(F):代表机器人原型机在研究者被动运动原型机移动臂时所产生的阻力;补偿力(4):为克服原型机系统内部阻力而产生的力;Kθ(t):原型机系统根据牵拉关节角度变化θ的产生的阻力变化.关节角度θ越大,原型机产生阻力越大;Bθ(t):原型机系统根据牵拉角速度θ模拟阻力变化,角速度θ越高,原型机产生阻力越大.教学仿真机器人原型机在每位评估者进行评定时运行随机化程序,随机呈现20次屈肘肌群Ashworth评级1、2、3、4级中的任意一项,以文本的形式在后台进行记录.评估者需要对机器人随机仿真模拟呈现的20次痉挛进行评定和记录,而后和机器人后台记录信息进行一致性分析.结果:原型机共随机呈现不同Ashworth评级共440次,1-4级分别呈现119、109、98、114次.使用R软件进行一致性分析,Ashworth 1-4级机器人仿真模拟和人工评定的一致率分别为:0.905、0.832、0.736、0.931,总体一致率为0.884.结论:教学机器人原型机对于卒中后典型痉挛仿真度较高,具备成为康复治疗学临床教学工具的潜能.

Objective: Contagious ecthyma is a severe and highly contagious disease caused by an orf virus (ORFV). The virus is responsible for substantial economic losses in the goat industry and threatens humans. We previously determined the role of ORFV129 protein, one of the five ankyrin-repeat proteins coded by the orf genome, in suppressing the transcription of pro-inflammatory cytokines IL-6, IL-1 & beta; and IFN-& gamma;. In the present study, we identified 14 cellular proteins (complement C1q binding protein [C1QBP], MCM7, EIF5A, PKM, SLC6A, TSPAN6, ATP6AP2, GPS1, MMADHC, HSPB6, SLC35B1, MTF1, P3H4, and IL15RA) that interact with ORFV129 using a yeast two-hybrid system in goat turbinate bone cells (GFTCs). The interaction between ORFV129 and (C1QBP), an immune-related protein, was confirmed using immunofluorescence co-localization and coimmunoprecipitation assays. C1QBP overexpression inhibited ORFV replication, whereas the knockdown of C1QBP promoted ORFV replication in GFTCs. Furthermore, ORFV or ORFV129 increased C1QBP expression in GFTCs, indicated that ORFV129-C1QBP interaction might contribute to the ORFV-induced host immune process. In addition, our research showed that ORFV increased the expression of ORFV129, cytokine IL-6, IL-1 & beta; and IFN-& gamma;. C1QBP overexpression induced IFN-& gamma; production and reduced IL-6 and IL-1 & beta; production. Conversely, C1QBP knockdown induced IL-1 & beta; production and reduced IFN-& gamma; and IL-1 & beta; production. Moreover, augmentation of ORFV129 expression enhanced the inhibition of the secretion of cytokines IL-6, IL-1 & beta;, and IFN-& gamma; induced by the altered expression of C1QBP. These findings suggest different downstream pathways might be involved in regulating different cytokines induced by ORFV129 expression in GFTCs.

为了解致病性大肠埃希菌在川西北高原牦牛中的分布特征及其致病性,本研究用PCR方法对细菌16S rRNA基因和毒力基因进行检测,并评价了牦牛源K99+F41十菌株的致病性.结果显示,分离的34个菌株生化鉴定均符合大肠埃希菌特征.所有菌16S rRNA基因与其他牛源大肠埃希菌同源性达99％以上.各菌株中毒力基因检出率分别为:hlyA100％、eaeA100％、K99 11.76％、F17 8.82％、F41 14.7％和 Stx1 5.88％;同时携带多种毒力基因的菌株分别为:K99+F41+hlyA+eaeA+8.8％、F17+hlyA+eaeA+8.8％、Stx1+hlyA+eaeA+5.9％、K99+F41+hlyA+2.9％、F41+hlyA+eaeA+2.9％.所有菌株包括O1、O72、O117、O126和O152等5种血清型,其中O152为优势血清型(44.1％).K99基因同源性分析显示,F2株与人源TEM160株、猪源WS株同源性最高;FR2株与牛源JE86-ST02株、E15042株同源性最高;FR3株与猪源202株同源性100％;H5-2与禽源HYulww1株同源性最高.系统进化树显示,FR2和FR3株与阿根廷牛源ETEC亲缘关系最近,可能存在相似的演化关联.K99+F41+菌株H5-2、F2、FR3、FR2对小鼠LD50分别为3.46× 108,4.53X 108,9.96× 107,5.29×108CFU/mL;死亡小鼠均出现肺脏充血水肿,肝脏和脾脏明显淤血变黑,肠壁变薄,肠腔内充满稀薄内容物等.本研究为川西北高原地区牦牛大肠埃希菌病防控提供了理论依据.

TSPAN6是四跨膜蛋白超家族成员之一,与病毒毒力、致病性及肿瘤发生相关.本研究旨在克隆山羊TSPAN6基因序列并进行生物信息学分析.以简州大耳羊为实验对象,无菌采集其肝脏,Trizol 法提取组织总RNA,并反转录为 cDNA,RT-PCR 技术克隆山羊TSPAN6基因序列,利用在线软件进行生物信息学分析.结果显示,成功克隆得到 TSPAN6基因,共 1400 bp(GenBank 登录号:ON337134),包括 5'UTR 149 bp,CDS 区长 738 bp,3'UTR 23 bp,编码 245 个氨基酸残基.山羊TSPAN6基因序列与绵羊、牛、猪、斑马、海豹和狐狸的相似性分别达到 99.32%、98.24%、93.63%、93.90%、92.41%和 92.68%.TSPAN6 蛋白分子式为 C1259H1952N310O346S18,分子质量为 27544.30 Da,是1种偏碱性、稳定的疏水性蛋白.TSPAN6 蛋白无信号肽,存在 4 个跨膜结构域,二级结构显示以 α螺旋为主,无β折叠结构,含有1个N糖基化位点与41个磷酸化位点.亚细胞定位预测 TSPAN6 蛋白主要定位于细胞膜,可能与 MAVS、ZFHX4、SEZ6L2 等 10 个蛋白存在相互作用.本实验成功克隆了山羊 TSPAN6基因,为后续山羊TSPAN6基因功能的研究提供了参考依据.

Porcine epidemic diarrhea virus (PEDV) is one of the critical pathogens causing diarrhea in piglets and has caused huge economic losses to the swine industry in worldwide. However, there is currently no effective therapeutic medication available for the treatment of PEDV. Natural compounds are a hot topic for researching and screening antiviral lead compounds due to their abundant sources, varied activities, and low toxicity. In this study, a total of 6 compounds from different plant sources were selected for in vitro anti-PEDV screening, including chrysin, naringenin, soy isoflavone, glycyrrhetinic acid, oleanolic acid, and geniposide. Then two active compounds, chrysin and naringenin, were further evaluated on PEDV infected cells at different stage. And the anti-PEDV mechanism was analyzed by molecule docking and molecular dynamics. The results showed that both chrysin and naringenin showed the most significant anti-PEDV activity by increasing the cell viability and decreasing the virus copy number. Both natural compounds could inhibit viral titer, mRNA and protein levels in the prophylactic and post-viral entry stages of PEDV infection. Furthermore, chrysin and naringenin mainly interacted with viral replicase proteins such as 3CLpro and PLP-2 through hydrogen bonds and hydrophobic forces. The complexes formed by chrysin and naringenin with the two PEDV replication proteases had high stability. These results suggested that chrysin and naringenin may exert antiviral effects by interacting with the virus 3CLpro protein or PLP2 protein, thereby affecting their role in the formation of PEDV non-structural proteins or interfering with virus replication. This study lays the foundation for developing chrysin and naringenin as novel anti-PEDV therapeutic drugs.

为了解引发四川某猪场腹泻疫情的猪轮状病毒(PoRV)基因组特征和遗传变异规律,采用RT-PCR分别扩增PoRV 11个基因节段,并克隆测序,通过生物信息学分析软件研究病毒基因组特征、变异性及遗传进化规律.结果显示,SCMY-1株基因合型为G3-P[13]-I5-R1-C1-M1-A8-N1-T1-E1-H1,即G3P[13]型毒株.该毒株VP2、NSP2和NSP4基因与人源毒株同源性最高,分别与3株人源轮状病毒RVA/Human-wt/LKA/R1207/2009株、RVA/Human-wt/ZAF/UFS-NGS-MRC-DPRU2291/2009和RVA/Human/CU331-NR/08株单独聚为一小支,推测这可能增加SCMY-1株跨种传播到人的风险.此外,SCMY-1株VP4基因与美国3个猪源毒株同处一个小分支;VP7基因与RVA/Pig/China/LNCY/2016株遗传进化距离最近.上述结果提示,PoRV可能在国际和国内生猪贸易过程中被广泛传播.与NCBI上已登录毒株相比,SCMY-1株VP4、VP7蛋白氨基酸分别存在13个和2个独有氨基酸位点的突变,可能对VP4和VP7两个主要抗原毒力及抗原性产生影响.重组分析显示,SCMY-1株VP3基因由两个猪源亲本株重组而来,推测这可能增强病毒毒力和宿主适应性.研究结果丰富了PoRV基因组学和分子流行病学相关研究资料,为PoRV预防控制提供理论参考,也为深入研究PoRV跨物种传播的分子基础提供科学依据.

[目的]了解中国西南部分地区流行的猪流行性腹泻病毒(PEDV)基因型及遗传变异规律.[方法]对2020-2021年四川和云南不同地区来源的200份腹泻猪肛门拭子进行RT-PCR检测,对PEDV S和ORF3基因进行克隆测序,利用Meg Align软件进行变异性分析,采用Mega 7.0软件进行系统进化分析,采用RDP4软件进行基因重组分析.[结果]在所有样本中PEDV总体阳性率为15.50％(31/200),群体阳性率为100％(8/8).检出的8株PEDV S基因和ORF3基因序列与已登录毒株相似性分别为90.5％～99.4％和92.9％～100％.对S基因的进化分析显示,8个PEDV流行株分布于G2a、G2b和G2c 3个亚群中.其中,四川SCWJSWUN02株与福建CH/FJXM/1/2012株遗传距离较近,云南YNKMSWUN01株与越南毒株单独聚为一支,表明当前西南地区PEDV的流行呈现出区域间传播的特征.与本地区近年流行毒株和常用疫苗株相比,其S蛋白氨基酸序列均存在不同程度的变异,但ORF3蛋白相对保守.部分毒株S蛋白存在连续氨基酸的插入或缺失,如在SCMYSWUN03株S蛋白59-62位氨基酸处发现4个氨基酸的缺失;在YNKMSWUN01株78-82位氨基酸处和84-88位氨基酸处分别有5个氨基酸插入和缺失.部分氨基酸突变位点存在于已鉴定的PEDV S蛋白受体结合中.此外,基因重组分析结果显示,SCMYSWUN03株可能发生了基因重组事件,概率为98.2％(P＜0.01).[结论]本研究发现当前西南地区流行的PEDV毒株包括G2a、G2b和G2c 3个亚群,毒株基因型具有多样性,且不同毒株间的变异性较大,丰富了 PEDV在四川和云南地区的分子流行病学调查资料,揭示了中国西南地区流行的8个PEDV毒株的分子遗传变异规律和进化特征,为本地区猪腹泻病防控措施的制定提供了理论依据.

为了建立一种快速鉴别PRRSV、基因2型CSFV、PRV和JEV的一步法多重RT-PCR检测方法,试验分别以PRRSV ORF6基因、基因2型CSFV E2基因、PRV gE基因和JEV E基因为靶基因构建重组核酸标准品;设计4对特异性检测引物,在同一反应体系中对One-step RT-PCR Enzyme Mix浓度、引物浓度和退火温度等进行优化,检验特异性、敏感性和重复性;用建立的一步法多重RT-PCR检测方法与商品化试剂盒同时对已知临床样本[35份PRRSV、45份CSFV、20份PRV(gE)和25份JEV阳性核酸和20份阴性核酸样本]进行检测,比较符合率.结果表明:经反应条件的优化确定一步法多重 RT-PCR 检测方法的最佳扩增体系[5×QIANGEN One-step RT-PCR Buffer 10μL,dNTP Mix 2 μL,One-step RT-PCR Enzyme Mix(20 U/μL)2 μL,上下游引物(基因 2 型 CSFV、JEV 引物浓度均为5 μmol/L,PRRSV引物浓度为3 μmol/L,PRV引物浓度为7 μmol/L)各1μL,模板1 μL,DEPC水补足至50μL]和扩增程序[50℃30 min;95℃15 min;94℃30 s,58℃30～45 s(基因2型CSFV、PRRSV 30 s,PRV、JEV 45 s),72℃1 min,共循环 35 次;72℃10 min].该方法对基因 1 型 CSFV 毒株(Shimen株)、CSFV疫苗株(HCLV株)、PRV疫苗株、BVDV及9种猪常见病毒和细菌病原均未检出;对PRRSV、基因2型CSFV、PRV和JEV核酸标准品检测下限分别为1.72×102 copies/μL、1.97x 103 copies/μL、1.94×103 copies/μL和2.08×102 copies/μL;该方法具有良好的重复性;与商品化试剂盒检测结果的符合率分别为PRRSV 100％(k=1)、基因2型CSFV 96.9％(k=0.93)、PRV 100％(k=1)和JEV 100％(k=1).说明本研究建立了一种能同时检测PRRSV、基因2型CSFV、PRV(gE基因)和JEV的快速、精准和高效的一步法多重RT-PCR检测方法,该方法具有良好的特异性、敏感性和重复性.

旨在研究鸭干扰素诱导的跨膜蛋白(duck interferon-induced transmembrane proteins,duIFITMs)和相关细胞因子在鸭甲型肝炎病毒3型(duck hepatitis A virus genotype 3,DHAV-3)感染早期的变化规律,以及duIFIT-Ms对DHAV-3的抑制作用,本研究对DHAV-3感染早期雏鸭肝中mRNA进行高通量测序分析,通过Real-time PCR验证duIFITMs及其相关细胞因子的变化;分别用pEGFP-duIFITM1和duIFITM1-siRNA构建duIFITM1过表达和敲减的鸭胚肝原代细胞(duck embryo liver cell,DELC)模型,通过该模型评价了duIFITM1对DHAV-3增殖的影响.结果显示:转录组测序分析及验证结果均表明,DHAV-3感染雏鸭后24和36 h duIFITM1显著上调(P＜0.01),IFITM5无明显变化.进一步用DHAV-3感染duIFITM1过表达和敲减的DELC细胞,与对照组和敲减组相比在过表达细胞中,在48、60 hDHAV-3拷贝数和病毒滴度均明显下降(P＜0.01).此外,经转录组测序分析共筛选出211个与抗DHAV-3免疫反应相关分子和74条显著富集的信号通路.经Real-time PCR检测,在攻毒后12和24 h肝中RIG-I和MDA5的表达水平与对照组差异不显著,但在36 h上调到对照组的4.23倍(P＜0.01)和3.61倍(P＜0.05),这与IFN-α/IFN-β早期表达滞后的趋势也恰好一致.同时,感染早期IRF1和IRF3的表达水平也显著上调.本研究首次探明duIFITM1对DHAV-3具有抑制作用,为以此开发新型抗病毒药物奠定了基础.对抗病毒感染早期的多个关键免疫分子表达变化规律的系统研究分析,可为DHAV-3感染与免疫的分子机制研究提供重要参考.

Hyaluronic acid-binding protein (HABP4) plays important roles in regulating cell cycle and apoptosis. However, its functions in regulating cell apoptosis remain unclear. To reveal the effects of HABP4 on cell proliferation, cell cycle and apoptosis, the HABP4 sequence was cloned, and we investigated the gain and loss functions of HABP4 in goat turbinate bone cells. Our results showed that a 1,496-bp HABP4 sequence was cloned successfully. The interference effect of siRNA1 on HABP4 was the strongest, reducing its mRNA expression level by 83%, decreasing the cells in the G0/G1 and S phases of the cell cycle and inhibiting cell growth and apoptosis. The overexpression of HABP4 produced contrasting results. Furthermore, an HABP4 knockdown caused the up-regulated expression of genes associated with apoptosis, including Bcl-2 and BCL2L11, but the down-regulation of Caspase3, Caspase7, Bax, PARP1, SOCS2 and P53 mRNA levels. Additionally, HABP4 overexpression significantly up-regulated the expression levels of Bax, Caspase3, Caspase7, BCL2L11, P53, SOCS2 and PARP1. However, the expression of Bcl-2 was down-regulated. These data provide an important foundation for further in-depth studies of HABP4 functions.

为了对四川省某肥育猪场疫病原因进行确诊并为其提供临床用药指导,研究从发病猪场采集病变组织样本进行细菌分离纯化;通过PCR分别检测并鉴定猪繁殖与呼吸综合征病毒(PRRSV)和副猪嗜血杆菌(HPS)基因型和血清型,并进行核苷酸变异性分析和遗传进化分析;用K-B纸片法筛选HPS敏感抗生素.根据病猪高热、耳根四肢发绀;全身淋巴结肿大,肺脏、肝脏肿大实变等主要临床症状和病理变化,结合PCR初步确定引发该次疫情的主要病毒为PRRSV.进一步对该毒株Nsp2基因进行克隆测序和比对分析发现其与广西分离株PRRSV HUN4-like GXBH1404株序列相似度高达99.17％.根据本次分离的毒株和参考的毒株建立了基于Nsp2基因的进化树,该毒株属于Lineage8谱系的HP-PRRSV毒株,与谱系8中的代表毒株JXA1和HUN4、GXBH1404及四川流行毒株SCCD16、SC2012等5个毒株相比,SCLZ202201株Nsp2基因共存在9个独有的碱基突变,与四川流行毒株相比共存在21个碱基突变,表明HP-PRRSV仍在四川地区持续流行,其基因多样性也在不断增加.经细菌分离纯化获得一株依赖NAD生长的革兰氏阴性短杆菌,结合PCR和生化鉴定,综合判定该菌为血清5型副猪嗜血杆菌(HPS)菌株.药敏试验结果显示,该细菌对新霉素、恩诺沙星、庆大霉素、头孢克洛、头孢氨苄较敏感;对林可霉素、卡那霉素、多西环素中介;对头孢拉定耐药.由此确定,该次猪场疫情由PRRSV HUN4-like毒株和HPS 5型菌株混合感染所致.研究确定了引发该次疫情的主要原因,为养殖场相关疫病的防控提供了理论依据.

高致病性猪繁殖与呼吸综合征(HP-PRRS)给我国养猪业带来了巨大的损失.为快速、便捷诊断该病,建立重组酶聚合酶扩增结合侧流层析技术(RPA-LFD)的HP-PRRS诊断方法.针对HP-PRRSV Nsp2基因特异序列,设计生物素(Biotin)标记的特异性引物和FAM荧光素标记的探针,对RPA-LFD的反应体系和条件进行优化,结果显示,HP-PRRSV RPA-LFD最佳反应体系为:上下游引物(0.42 pmol/μL)各2.1μL,探针(0.3 pmol/μL)0.6μL,Rehydration buffer 29.5μL,cDNA模板1μL,ddH2 O 12.2μL,MgOAc 2.5μL,共50μL;最佳反应条件为:37℃,20 min.该方法同时检测高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)、猪繁殖与呼吸综合征病毒VR2332株(PRRSV VR2332)和NADC30-like株等10种常见感染猪的病原,结果显示,该方法除对HP-PRRSV检测呈阳性外,对PRRSV VR2332株、NADC30-like和其它9种感染猪的病原检测均为阴性,特异性强;该方法对10倍系列稀释的质粒标准品检测结果显示,该方法对质粒标准品的最低检测限为4.05×101拷贝/μL,灵敏度高;该方法与HP-PRRSV商品化荧光检测试剂盒同时检测50份临床样品,二者符合率为100％.本研究首次建立了一种特异、灵敏、方便、快速、现场化的HP-PRRSV RPA-LFD检测方法,为HP-PRRS的快速诊断和流行病学调查提供了新的技术手段.

旨在探究羊口疮病毒ORFV118蛋白对山羊睾丸支持细胞周期、凋亡以及诱导机体免疫应答相关的4种细胞因子(IL-1β、IL-6、IFN-γ和TNF-α)表达水平的影响.在ORFV118基因克隆基础上,成功构建真核表达载体pEGFP-ORFV118;将pEGFP-ORFV118转染山羊睾丸支持细胞,Western Blot鉴定ORFV118蛋白表达的正确性;流式细胞仪检测ORFV118基因对细胞周期及凋亡的影响;RT-qPCR检测细胞周期相关基因CDK2和P21、凋亡相关基因Bax、Bcl-2、Caspase3、Caspase7、P53和BCL2L11的mRNA的表达水平,并利用RT-qPCR和ELISA方法检测免疫相关的细胞因子表达的变化.结果显示,成功扩增ORFV118基因,全长为309 bp.ORFV118蛋白的表达阻滞了细胞的DNA合成期(S期),促进了DNA合成后期(G2/M期)且下调基因CDK2的mRNA表达水平;可抑制细胞的凋亡作用,且下调促凋亡基因Caspase3、Caspase7、SOCS2的mRNA表达水平;对细胞因子IL-1β和IFN-γ呈不同程度的抑制作用,而对IL-6和TNF-α呈促进作用.

旨在了解四川某规模化养殖场羊源大肠埃希氏菌的耐药表型及耐药基因的携带情况.收集50份简州大耳羊源小肠内容物,采用细菌学方法对大肠埃希氏菌进行分离鉴定;采用K-B纸片法评价分离菌对常用抗菌药物的敏感性;对细菌携带的β-内酰胺类、氨基糖苷类、四环素类、喹诺酮类耐药基因的携带情况进行分析.结果显示,从50份样本中分离鉴定出7株羊源性大肠埃希氏菌;药敏试验及多重耐药试验结果显示,7株大肠埃希氏菌对阿莫西林表现出全部耐药;对氨苄西林、四环素、链霉素的耐药率分别为42.86％、71.42％和28.57％,且表现了一定的多重耐药性,其中3耐占42.86％.7株分离株对β-内酰胺类blaTEM-1基因检测的携带率为100.00％,氨基糖苷类rmtB、rmtC、rmtD基因携带率均为14.29％,四环素类Tet(A)、Tet(C)、Tet(D)、Tet(E)、Tet(M)基因携带率分别为71.43％、14.29％、42.86％和42.86％、42.86％,喹诺酮类qnrD、qnrS、oqxA基因携带率分别为57.14％、71.43％和71.43％.研究结果表明,7株羊源大肠埃希氏菌均表现出一定的耐药性,部分耐药基因携带率较高,为该场临床预防用药提供一定的参考.

为了解近年来四川地区基因C型鸭甲肝病毒(DHAV-C)流行株的生物学特性、基因组特征和遗传变异规律,本实验采用RT-PCR法筛选410份四川不同地区DHAV-C的阳性肝脏样品,结果显示,DHAV-C在四川省广泛流行,部分养殖场阳性率高达64.62％(42/65).以鸭胚尿囊腔接种法分离DHAV-C并测定其半数致死量(ELD50),同时观察病毒对鸭胚肝原代细胞(DELC)的适应性,并利用透射电镜观察病毒形态.结果显示,分离获得4个病毒株SWUNC1、SWUNC2、SWUNC3和SWUNC4,ELD50分别为:10-4.5/0.2 mL、10-5.33/0.2 mL、10-5.33/0.2 mL和10-7.65/0.2 mL.其中仅SWUNC4株可以引起DELC明显的CPE,在透射电镜下观察到细胞质中圆形的病毒粒子.对分离的4株DHAV-C进行分段克隆后拼接全基因组序列,分别对其进一步测序并分析.结果显示,4个分离株之间的同源性为96.9％～99.2％,与NCBI中已登录的41株病毒相比同源性为92.7％～99.1％.构建的VP1基因进化树结果显示4个分离株均属于GⅡ亚型,且与四川往年流行株距离较远,提示近年来四川流行的DHAV-C基因组可能出现变异.进一步和国内外代表株比较,4个分离株的VP1基因均存在独有氨基酸突变位点,其中SWUNC4株VP1基因突变最多为7个.4株分离株与四川早期分离株的抗原指数有明显差异,在SWUNC4株VP1蛋白aa101～aa111处还出现一个新的B细胞预测表位,提示传统疫苗株可能对分离株的保护效果下降.本研究丰富了DHAV-C基因组学研究资料,揭示了分离株遗传变异规律,也为DHAV-C检测靶点和疫苗候选株的筛选奠定了基础.

猪冠状病毒是猪病毒性腹泻的一类重要病原,感染仔猪死亡率可达100％,给养猪业带来了巨大的经济损失.文章将对猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪德尔塔冠状病毒以及猪急性腹泻综合征冠状病毒等4种猪冠状病毒检测方法研究现状进行总结,对各种方法的优缺点和应用前景进行系统的讨论和展望,以期为临床上猪冠状病毒的高效检测、精准防控,以及未来猪冠状病毒新型检测方法的研究提供新思路.

研究旨在建立一种猪伪狂犬病病毒(PRV)的探针法荧光定量检测方法.选用PRV gE基因为靶基因,比对不同野毒在该基因的保守区域设计特异性引物和探针,构建PRV gE基因片段的重组质粒作为标准品,对该方法的反应体系和条件进行优化,并进一步评估该方法的敏感性、特异性和稳定性,最后与商品化试剂盒同时检测临床样本,比较符合率.结果表明,该研究建立的TaqMan荧光定量PCR检测方法的最佳反应条件为上下游引物和探针量分别为0.2μmol/L和0.05μmol/L,最佳退火温度为58℃.该方法具有良好的特异性,对PRVBartha-k61疫苗株、猪流行性腹泻病毒(PEDV)、猪细小病毒(PPV)、猪A群轮状病毒(PoRV)、猪德尔塔冠状病毒(PDCoV)、猪传染性胃肠炎病毒(TGEV)、猪链球菌(S.suis)、猪源大肠杆菌(E.coli)、猪源巴氏杆菌(P.multocida)、猪霍乱沙门氏菌(S.choleraesuis)和葡萄球菌(S.aureus)等11个菌毒种均无非特异性扩增.该方法的最低检测限为3.92 copies/μL.重复性试验结果显示,组内和组间变异系数均≤2％.与商品化的荧光定量PCR检测试剂盒比较,符合率均为100％(n=50).综上所述,该研究建立了一种灵敏高效的检测PRV野毒株的TaqMan荧光定量PCR方法,该方法可以成为猪伪狂犬病早期快速诊断和流行病学调查的一种可靠的技术手段.