本试验选用镧系元素铕(Eu)为荧光物质标记布鲁氏菌脂多糖抗原(LPS),建立了布鲁氏菌抗体高敏荧光免疫层析快速检测试剂盒(Bru-LFICA).经相关试验验证,得出该试剂盒特异性强,敏感性高,重复性好,符合率高,且兼具操作方便,检测快速(15min),价廉,不需特殊设备和专业技术人员等优点,特别适合于基层兽医站、养殖场的即时快速检测.

兔病毒性出血症2型(RHD2)是由兔出血症病毒2型(RHDV2)引起的家兔和野兔的高度传染性、急性致死性疫病.RHDV2目前主要在欧洲流行,引起的临床症状与经典兔病毒性出血症(RHD)相似,但毒株特性差异较大.2020年4月四川省两个兔场的家兔同时出现了疑似RHD症状,但用经典RHDV疫苗免疫后家兔发病及死亡情况均无好转.分别采集两个兔场的病死兔组织样品进行实验室诊断,使用OIE推荐的RHDV2特异性引物进行RT-PCR检测,两个发病兔场的兔组织混合样品均扩增出大小约800bp的片段.进一步的测序和Blast比对分析发现来自两个发病兔场的2条序列均与RHDV2参考毒株的VP60部分序列高度同源,其中同源性最高的是荷兰RHDV2分离株RHDV2-NL2016,分别为98.01％和97.95％.分析结果表明,报告发病的两兔场的病死兔均为RHDV2感染,这是我国首次官方确诊RHD2疫情.分别将两个兔场组织病料中的RHDV2毒株命名为SCADC-JT01和SCADC-JT02,有关这2株新毒株的毒株特性和基因组特征还有待进一步研究.

本试验以镧系元素铕(Eu)为荧光标记,用纯化的PRV-gE和PRV-gB表达蛋白抗原建立了检测猪伪狂犬病gE、gB抗体的镧系荧光免疫层析快检试剂盒.经特异性、敏感性、重复性试验及与同类产品的对比试验,得出该试剂盒特异性强、敏感性高、重复性好、符合率高,且质量稳定,保质期长,是实现猪伪狂犬病即时现场快速检测的一种血清学检测方法.

本试验以镧系元素铕(Eu)为荧光标记建立了检测猪蓝耳病病毒抗体的镧系荧光免疫层析快检试剂盒,经特异性、敏感性、重复性试验及与同类产品的比对试验,表明该试剂盒特异性强、敏感性高、重复性好、符合率高;且质量稳定,保质期长,是实现猪蓝耳病即时现场快速检测的简单而实用的血清学检测方法.

本试验以镧系元素铕(Eu)为荧光标记,用禽流感通用型和H7亚型单克隆抗体,研制了检测禽流感抗原的镧系高敏荧光免疫分析快检试剂盒(LFICA),经特异性、敏感性、重复性试验及与同类产品的对比试验,表明该快检试剂盒(LFICA)特异性强,敏感性高,重复性好,是目前禽流感病原快速诊断初筛和流行病学调查最快捷、简单和实用的诊断检测方法.

Transforming growth factor beta 1 (TGF-β1) was of importance in the pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV). To determine whether knockdown of TGF-β1 gene expression could facilitate the control of PRRSV infection, specific sequences for expressing shRNA targeted to porcine TGF-β1 gene were synthesized and cloned into pSilencer 3.1-H1 neovector. Then they were used to transfect peripheral blood mononuclear cells of Tibetan pig (Tp-PBMCs) followed by PRRSV inoculation. The positive recombinant plasmids were screened for inhibition of TGF-β1 gene expression by real-time quantitative RT-PCR. Conversely, the mRNA level of PRRSV in shRNA treated Tp-PBMCs dramatically decreased, and there were significant increases of the transcription of immune genes, such as interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-alpha (IFN-α), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), toll-like receptor 3 (TLR3), toll-like receptor 7 (TLR7), Myeloid differentiation primary response gene (88) (MyD88), and interleukin-27p28 (IL-27p28). However, the expressions of IL-8 and IL-10 genes significantly reduced in comparison to the control infected cells. In addition, transfection with the shRNA plasmids significantly elevated the viability of immune cells. Therefore the knockdown of TGF-β1 gene expression by shRNA not only inhibits the replication of PRRSV but also improves immune responsiveness following viral infection, suggesting a novel way to facilitate the control of PRRSV infection in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is a severe, economically important infectious disease of pigs.Current vaccines to control the disease, however, are inadequate due to low immunity;and the present study was conducted to explore an effective adjuvant to enhance the immunological efficacy.A recombinant plasmid containing the porcine IL-23 gene was encapsulated in chitosan nanoparticles and Pluronic-L64 designated as VRIL23-CNP-Pluronic, which was tested following intramuscular injection of 28 day-old mice in comparison with VR1020-Pluronic as the control.Over a 35 day observation period, PRRSV-specific antibody, IgG1 and IgG2 a titers, CD4+ and CD8+ T cells increased significantly in the treated mice (P<0.01).As analyzed by qRT-PCR, expression levels of IL-23, TLR1, TLR6, STAT1, IL-10, TNF-α, IL-15 and CD62 L genes were also significantly up-regulated in comparison with those of control mice (P<0.01).These results show that co-injection of VRIL23-CNP-Pluronic with PRRS vaccine enhances both innate and adaptive immunity of mice, and provide support for its development as a novel vaccine adjuvant for control of swine PRRS.

细菌性传染病是生猪养殖业的严重威胁之一,近年来其发病情况呈现出新变化,对动物产品质量和公共卫生安全造成严重危害.对病原进行快速、准确的检测,是预防和控制猪病的有效手段,对养猪业发展和人畜健康具有重要意义.伴随生命科学技术的发展和学科间的交叉融合,传统的病原菌分离培养、普通PCR方法和早期免疫学等检测技术均朝着精确高效的方向不断发展革新,并以传统方法为基础建立了多种新型检测技术.文章重点介绍了猪细菌性传染病病原检测的几种新技术:以核酸分子生物学为基础的实时荧光定量PCR法(qPCR)、环介导等温扩增法(LAMP)、夹心DNA杂交(DNAH)等;以免疫学为基础的免疫胶体金标记技术、时间分辨荧光免疫分析法(TRFIA)、免疫磁珠分离法(IMS)等;以蛋白质分子生物学为基础的细菌质谱鉴定技术;以及适用于现场快速诊断的即时检测(POCT)技术,并分析了这几种新技术的优缺点,为不同条件的检测机构和研究部门在开展猪细菌性传染病检测和研究时选择相应的方法提供一定的参考,以期在猪病预防控制中发挥更加积极的作用.

本试验以镧系元素铕(Eu)为荧光标记,用纯化的PCV-2表达蛋白抗原建立了检测猪圆环病毒病抗体的镧系荧光免疫层析快检试剂盒.经特异性、敏感性、重复性试验及与同类产品的对比试验,得出该试剂盒特异性强、敏感性高、重复性好、符合率高,且质量稳定,保质期长,是实现猪圆环病毒病即时现场快速检测的简单而实用的血清学检测方法.

为了分析比较不同的商品化检测方法在禽流感病毒快速诊断中的实用性,本试验运用禽流感高敏快速荧光免疫法、胶体金免疫层析纸条法和实时荧光RT-PCR三种方法同时对禽流感病毒H5、H7和H9三个亚型抗原各12个稀释度的样本进行检测.结果 表明,禽流感高敏快速荧光免疫法对三种亚型抗原的灵敏度较高,各亚型检出最低稀释度分别为H5:1∶2560,H71∶1 280,H9:1∶640,该方法快捷简便,且设备便携,通过云平台处理数据,适用于现场即时检测(POCT)等;进口胶体金免疫层析纸条法对H5、H9亚型抗原的灵敏度高,对H7亚型抗原的灵敏度较低,且国内产品存在假阳性、准确性较低等缺点,但均操作简单,适用于基层初筛;实时荧光RT-PCR法对三种亚型抗原的灵敏度高,稀释度均可达1∶40960以上,但耗时长,设备和技术要求相对较高,更适用于实验室确诊.

为开发高效安全经济的免疫调节剂,本研究构建了共表达猪IL-4/6和猪抗菌肽融合基因VRP的重组毕赤酵母SG46P.通过猪淋巴细胞增殖试验和抑菌试验检测其发酵上清液的生物学活性和抑菌活性,并用该重组毕赤酵母对30只21日龄雌性ICR小鼠进行灌胃接种.接种后,每周采集小鼠尾部静脉血进行免疫功能分析试验;并在第28 d用大肠杆菌和金黄色葡萄球菌进行小鼠腹腔攻毒试验.结果显示:实验组SG46P的发酵上清液较对照组能显著刺激猪淋巴细胞的增殖(p＜0.05),且该发酵上清液具有明显的抑菌作用(p＜0.05).接种重组毕赤酵母SG46P的小鼠的体质量有所增加(p＞0.05)且其外周血中白细胞数量、血红蛋白含量均显著高于对照组(p＜0.05);其血清中IgG、IgG1和IgG2a抗体水平均较对照组显著增加(p＜0.05),其外周血中CD4+T和CD8+T淋巴细胞数量均显著高于对照组(p＜0.05);TLR4、TLR9、IL-2、IFN-γ、IL-4、IL-6、CD62L、IL-7、IL-23、CAMP和Crp4基因的转录水平均显著高于对照组(p＜0.05).攻毒后,接种SG46P的小鼠的生存率显著高于对照组(p＜0.05).结果表明:重组毕赤酵母SG46P不仅能够显著提高小鼠免疫基因的转录水平,有效提高动物的先天和获得性免疫水平,还可明显增强小鼠抗感染能力,这为研制高效安全经济的分子免疫调节剂和防治动物传染病开拓了新途径.

本研究以纯化的猪瘟病毒(CSFV)重组E2蛋白和纯化全病毒为抗原,用镧系元素铕(Eu)为荧光标记,建立了猪瘟抗体镧系荧光免疫层析快检法,经特异性、敏感性、重复性试验,表明该方法具有敏感性和特异性强、重复性好、快速、无辐射、高通量等优点,且操作简单、省时省力,该方法完全可以用于CSF抗体检测和免疫效价评估.

为了研发安全有效和增强动物免疫力的新生物制剂,本实验以共表达猪IL-4/6与猪抗菌肽基因和猪IL-2与IL-4/6基因的两种重组毕赤酵母菌进行高密度发酵,并拌入饲料饲喂45日龄仔猪(分组为A1、A2和C组).实验结果表明,在0～42d内,A1和A2实验组较对照组仔猪体重显著增加(P<0.05),CD8+T细胞数量、特异性抗猪瘟和蓝耳病抗体显著增多(P<0.05),实验组的TLR-2、4、7、NF-kB(p50)、Bcl-2、CD45和CD62L等免疫相关基因表达水平也明显升高(P<0.05).此外,实验后期,A2组的前述免疫相关基因表达水平明显高于A1组和C组(P<0.05).这些结果证明,A1和A2组免疫生物制剂能较好地促进仔猪的生长,提高其先天和获得性免疫机能,A2制剂的免疫调节生物效应较优,有望进一步研发为新型安全的免疫生物制剂,为动物的疾病防控提供新技术支持.

Objective In order to develop a novel effective immunomodulator to enhance pig resistance against post-weaning muhisystemic wasting syndrome,we used the recombinant plasmid co-expressing the pig interleukin-2 (IL-2) and fusion interleukin-4/6 (IL-4/6) proteins which we constructed before,and studied its effect on immune response of piglets.Methods The recombinant plasmid was first encapsulated in chitosan nanoparticles by the ionotropic gelation method,and generated VRIL-4/6-2-CS.Then piglets were divided into 2 groups and intramuscularly injected with VRIL-4/6-2-CS or saline followed by the injection of pig circovirus-2 (PCV-2) vaccine,respectively.The blood was collected from each piglet on days 0,7,14 and 28 to assay the immunological changes.Results The number of IgG2a,CD4+,CD8+ T cells increased significantly in the sera or blood of piglets that treated with VRIL-4/6-2-CS (P ＜ 0.05).Furthermore,the expression levels of IL-2,IL-4,IL-6,TNF-α,TLRs (TLR-2,7) and STATs (STAT-1,2,3) genes elevated significantly in the piglets that treated with VRIL-4/6-2-CS (P ＜ 0.05).Although no significant differences were observed in the levels of PCV-2-specific antibody,the growth performance of VRIL-4/6-2-CS treated piglets was remarkably improved in comparison with that of control (P ＜ 0.05).Conclusion VRIL-4/6-2 entrapped in chitosan is a promising effective adjuvant to promote the immune responses of pig vaccinated with PCV-2.

试验发酵制备融合表达猪IL-2基因与IL-4、6基因、融合表达猪IL-4/6基因和牛源抗菌肽的两种重组毕赤酵母,分别用以饲喂仔猪,每2d饲喂1次,连续饲喂4周.在试验的第0、14、28和42天,测定各组仔猪的总体重及饲料消耗量,在试验的第0、7、14、28和42天,采取仔猪前腔静脉血,测定外周血免疫细胞数量变化、Th和Tc细胞数量变化、特异性抗体变化以及免疫应答相关基因表达水平的变化,以探讨2种免疫制剂对仔猪生长与免疫方面的调节效应.结果发现,在试验后第42天,A1、A2试验组仔猪平均增重量分别高于对照组C组3.47 kg和1.66 kg(P＜0.05).试验组血样中白细胞、CD8+T细胞、CSF特异性抗体和PRRS特异性抗体含量相比于对照组C明显增加(P＜0.05).此外,A1、A2组的免疫相关基因(TLR2、TLR4、TLR7、TLR9、STAT1、STAT3、NF-kB、IFN-、CD45和CD62L)的表达水平较C组显著升高(P＜0.05).总的来说,A1和A2两种免疫制剂,既能有效促进仔猪生长和发育,又能增强仔猪免疫应答水平,可进一步开发为免疫调节剂.

Increase of interleukin-10 (IL-10) induced by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection has been intensely studied to inhibit the anti-viral responses of host pigs. Blockade of expression of IL-10 receptor (IL-10R) by RNA interference (RNAi) may relieve the immunosuppression caused by excessive IL-10 in PRRSV infection. The recombinant short hairpin expressing plasmid targeted to pig IL-10Rα was transfected into peripheral blood mononuclear cells of Tibetan pig (Tp-PBMCs) prior to PRRSV inoculation, then the replication of PRRSV and immune responses in Tp-PBMCs were evaluated. The recombinant interfering plasmid greatly decreased PRRSV yield. The transcriptional level of IL-10Rαwas obviously inhibited by recombinant interfering plasmid; the expression of IL-10 was also down-regulated, while that of TGF-β1 was not affected. Furthermore, the recombinant plasmid notably up-regulated the mRNA levels of TLR3, TLR7, IFN-α, IFN-γ, IL-2, IL-4, IL-12p40 and MyD88, while that of IL-8 was apparently decreased; In addition, cell viability of Tp-PBMCs was clearly enhanced by the interfering recombinant plasmid. Our results suggest that knockdown the expression of pig IL-10Rα can evidently inhibit the PRRSV infection and enhance the anti-viral immune responses of pig immune cells, which may be a promising way for preventing virus infection and developing new effective immune-regulator to strengthen the host immunity against PRRS.

In order to develop a secure and competent technique to express the human immune gene for fighting infections, we cloned and expressed the BD2/3 using VR1020 (a eukaryotic expression plasmid). BD2/3 contains human β-defensin 2 (BD2) and human BD3. To explore safe and effective DNA delivery molecules in vitro and in vivo, the fusion genes of BD2/3 were used as an immune-labelled gene to verify transfection effectivness of modified chitosan (CS). Plasmid of VR1020-BD2/3 was packed with biomaterials: CS, average molecular weight: 25000D; polyethylene glycol-O-chitosan-polyethylenimine (PEG-O-CS-PEI); liposomes (LP); polyamine cationic liposomes (PCL); polyamine cationic liposomes of protamine (PCL-protamine) by ionotropic gelation. We observed that BD2/3 fusion gene showed high bioactivity in vitro and in vivo. The BD2/3 fusion protein inhibited the proliferation of bacteria (S. aureus, S. pneumoniae, P. aeruginosa and E. coli). The Kunming mice were immune to these nanoparticles and we analyzed their delivery efficiency and gene expression effect. BD2/3 results in multiple changes of innate and required immune system of mice. BD2/3 increases expression of IgG, IgG1, IgG2a, IL-2, IL-6, IFN-γ, as well as of lymphocytes and monocytes. Following challenge with virulent E. coli, CD4+ and CD8+ positive T-cell counts were highly elevated in the BD2/3 immunized mice, resulting in higher survival rates of mice. These results indicate that nanoparticles containing modified CS and BD2/3 are potentially safe and effective drugs in vivo to improve the immunity against bacterial infection and enhance innate immunity and adaptive immunity against infectious diseases.

本研究采用壳聚糖及其改性分子离子交联法包裹制备β-内啡肽基因重组质粒纳米颗粒,然后肌肉注射小鼠,另设口服重组酵母菌组,研究β-内啡肽基因在小鼠体内表达的生物效应.结果发现:肌注质粒与口服酵母菌均可促进小鼠生长,增加小鼠体重;外周血中CD4+T、CD8+T细胞数量以及小鼠血清IgG含量均显著提高(P<0.05),免疫相关基因的相对表达量也都显著提高(P<0.05);试验组小鼠血清中的β-内啡肽含量较对照组明显增加(P<0.05).表明β-内啡肽基因重组质粒壳聚糖纳米颗粒能有效调节小鼠免疫反应,增强机体抗应激的能力.

以2A自剪接技术构建猪白细胞介素4/6与牛融合抗菌肽(FBC)共表达重组毕赤酵母,为研究重组酵母对小鼠体内外的免疫协同效应,首先采用猪淋巴细胞增殖试验和抑菌试验研究重组毕赤酵母的体外生物学活性;再以发酵的重组酵母菌液进行小鼠灌胃试验,在试验的28 d用致病性大肠杆菌和金黄色葡萄球菌进行腹腔攻毒,每周采血检测小鼠的生长和免疫功能变化.体外试验结果显示,试验组比对照组显著刺激猪淋巴细胞增殖;明显抑制大肠杆菌标准菌、大肠杆菌耐药菌、金黄色葡萄球菌标准菌和金黄色葡萄球菌耐药菌的生长(P＜0.05).小鼠灌胃试验结果表明,试验组小鼠外周血的白细胞、Th和Tc细胞含量显著多于对照组;试验组tlrs (tlr-1,4,6,9),il-1,il-2,il-4,il-6,il-7,il-15,il-23和cd62l基因的表达明显高于对照组;试验组血清中的IgG、IgG1和IgG2a的含量比对照组显著增加.此外,试验组小鼠的体质量比对照组明显增加.攻毒后试验组80％的小鼠存活,而对照组小鼠则有明显的炎症及临床症状,最后全部死于感染,这表明试验组的存活率显著高于对照组(P＜0.05).白细胞介素4/6与牛融合抗菌肽共表达重组毕赤酵母能够有效提高小鼠免疫功能,为探索研制增强动物免疫机能的安全高效的免疫调节剂提供新途径.

为了开发经济实用的新型免疫调节剂,以2A自剪切技术构建重组毕赤酵母Pichiapaaoris共同表达猪白细胞介素-2(IL-2)和融合抗菌肽基因,然后以重组基因工程酵母菌饲喂ICR小鼠,以实时荧光定量PCR、ELISA和流式细胞计数评估其对小鼠免疫力和生长的影响.实验结果显示,处理组(融合抗菌肽重组酵母菌、IL-2和融合抗菌肽重组酵母菌)小鼠的生长明显优于对照组(空质粒酵母菌)(P＜0.05),且血清IgG、IgG1和IgG2a含量显著升高(P ＜0.05);TLR1、TLR4、TLR6、TLR9、IL-7、IL-15、IL-23、CD62L、IL-2、IL-4、IL-6、IL-12、CRP4和CAMP基因的表达水平显著增加(P＜0.05).同时,处理组小鼠血液白细胞Th和Tc细胞明显增多(P＜0.05),免疫力和存活率均明显高于对照组(P＜0.05).重组酵母菌能有效增强小鼠的先天和获得性免疫力,促进生长,可进一步开发成安全有效的免疫调节生物制剂,改善动物的生长和免疫力.