IntroductionAutophagy is an important process in host cell responses to viral replication and spread, including those against infectious bursal disease virus (IBDV). Programmed death-1 (PD-1) is a known immunoinhibitory receptor, and its expression causes immune dysfunction in B lymphocytes, resulting in increased progression of immunosuppressive diseases. However, the role of PD-1 in autophagy during IBDV infection remains unclear.MethodsWe investigated the mechanism by which chicken PD-1 regulates autophagy during IBDV infection.ResultsIBDV infection enhanced PD-1 expression in chicken tissues and DT-40 cells. Subsequent interaction analyses revealed that PD-1 interacted only with the viral protein VP2 to enhance the IBDV replication in DT-40 cells. PD-1 overexpression significantly increased IBDV-induced autophagy, whereas silencing of PD-1 had the opposite effect in IBDV-infected DT-40 cells. Furthermore, PD-1 enhanced the activation of FoxO1 via the PI3K/AKT pathway. Finally, we demonstrated that autophagy is critical for role of PD-1 in regulating VP2 protein expression and IBDV titers.DiscussionThese findings present a novel mechanism wherein PD-1 induces autophagy by activating the PI3K/AKT/FoxO1 pathway to facilitate IBDV replication, providing a new avenue in developing universal vaccine adjuvants for IBDV infection control.

Breakthrough progress has been made in the molecular mechanism research and clinical application of PD-1/PD-L1 in the regulation of immunosuppression and tolerance mainly in human and mouse fields, but is relatively slow in other species. The eukaryotic expression vectors pECFP-Fc-1 and pEYFP-Fc-L1 for high expression of canine PD-1 and PD-L1 proteins were constructed and transfected into human embryonic kidney 293 T cells. Fluorescence microscopy, laser scanning confocal microscopy, and immunofluorescence technology were used to identify the expression and membrane localization of the target proteins in human embryonic kidney 293 T cells. The binding activity of the target proteins expressed on the model cell was identified by eukaryotic expression vector co-transfection and immunocoprecipitation. The results showed that canine PD-1 and PD-L1 proteins were expressed on the membrane surfaces of their respective positively transfected cells. The cell membrane complex was further analyzed by co-immunoprecipitation technology, PD-L1 protein components were successfully detected in the pull-down complex of canine PD-1 antibody, and the two target proteins expressed in the model cells showed good mutual binding activity. Further research is needed to evaluate high throughput and a reliable method for screening drugs that block the PD-1 and PD-L1 pathway.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a highly contagious disease with high morbidity and mortality that affects the global swine industry. Despite efforts to control it, there is still a widespread dissemination of PRRSV with obvious genetic variations in swine population, resulting in huge economic losses annually. Consequently, accurate laboratory diagnosis is crucial for the rapid confirmation of PRRSV infections. An immunoperoxidase monolayer assay (IPMA) was developed for the specific and sensitive detection of PRRSV based on a broad-spectrum anti-PRRSV monoclonal antibody (mAb) 28F6. The mAb 28F6-based IPMA could specifically detect PRRSV and possessed no cross-reactions with CSFV, PCV2, and PEDV. Sensitivity analysis showed that the limit of detection of the IPMA reached 10− 2.25 TCID50/100 µL. There was no significant difference in the detection of PRRSV of different passages with different batches of mAb 28F6, indicating that the IPMA had excellent repeatability. Additionally, the IPMA was capable of detecting multiple PRRSV variants, including field strains (e.g., BJ-4, HN07-1, and HNhx) and vaccine strains (e.g., HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98). Validation of the IPMA with qRT-PCR showed 100

Background Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a highly contagious disease with high morbidity and mortality that affects the global swine industry. So far, there is still a widespread dissemination of PRRSV with obvious genetic variations in swine population, resulting in huge economic losses annually. Therefore, accurate laboratory diagnosis is needed to quickly confirm PRRSV infection. Results An immunoperoxidase monolayer assay (IPMA) was developed for the specific and sensitive detection of PRRSV based on a broad-spectrum anti-PRRSV monoclonal antibody (mAb) 28F6. The mAb 28F6-based IPMA could specifically detect PRRSV and possessed no cross-reactions with CSFV, PCV2, and PEDV. Sensitivity analysis showed that the limit of detection of the IPMA reached 10− 2.25 TCID50/100 µL. There was no significant difference in the detection of PRRSV of different passages with different batches of mAb 28F6, indicating that the IPMA had good repeatability. In addition, the IPMA could recognize a number of PRRSV variants including field strains such as BJ-4, HN07-1, and NADC30-like strain, as well as vaccine strains like HuN4-F112, JXA1-R, TJM-F92, GDr180, VR2332, CH-1R, and R98. Validation of the IPMA showed that it was in 100% consistency with qRT-PCR on the detection of 108 clinical samples. Conclusions The IPMA could meet the demand for the specific and sensitive detection of PRRSV, which is helpful for accurate monitoring and early warning of PRRSV infections.

Programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) binding contributes to immune evasion mechanisms responsible for B lymphocyte exhaustion and apoptosis. This facilitates immunosuppression in chronic viral infections, including infectious bursal disease virus (IBDV). Our previous study showed that PD-1 and PD-L1 expression increases in the peripheral blood mononuclear cells of chickens infected with IBDV. However, due to their high production costs and immune-related adverse events, monoclonal antibodies targeting PD-1 or PD-L1 are unsuitable therapeutic agents. Thus, in the current study, we designed peptides with optimized binding sites for PD-1 and investigated their ability to disrupt PD-1/PD-L1 binding and restore B lymphocyte function in vitro. The peptide gCK-16 exhibited a high affinity for PD-1 (KD: 3.37 nM) and effectively inhibited the PD-1/PD-L1 interaction in vitro. Moreover, gCK-16 significantly enhanced B lymphocyte proliferation. Remarkably, gCK-16 treatment abrogated the IBDV-induced upregulation of PD-1/PD-L1, NF-κB activation, and B lymphocyte apoptosis. Additionally, IBDV infection attenuated PI3K/AKT pathway activation in B lymphocytes, while gCK-16 treatment increased immunoglobulin M (IgM) production in IBDV-infected B lymphocytes. Together, these results demonstrate that gCK-16 treatment can potentially enhance B lymphocyte function against IBDV infection, guiding the development of vaccine adjuvants to effectively prevent IBDV-induced avian immunosuppression.

文章分析了动物生理学虚拟仿真实验在生物技术专业学生培养中的重要性.首先介绍了动物生理学虚拟仿真技术的特点及其优势;然后结合实例阐述了动物生理学虚拟仿真实验在生物技术专业学生培养中的应用,包括提高学生的实验操作能力、深化学生对生理过程的理解,以及培养学生的科学研究思维能力等;最后指出了动物生理学虚拟仿真实验在生物技术专业学生培养中存在的不足和未来的发展方向.文章为教育教学改革提供了思路和借鉴,同时为动物生理学虚拟仿真实验技术的发展进行了一个全面且深入的分析.

To establish an indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)method for the detection of zilpterol(ZIL)residues,in this study,based on the obtained rabbit anti-ZIL polyclonal an-tibody serum and the prepared complete antigen as the coating source,an ic-ELISA method for the detec-tion of ZIL residues was established by optimizing the reaction conditions;The results showed that the best dilution ratio of rabbit serum was 1:6 000,and the best antigen coating concentration was 2 μg/mL,the half inhibition concentration(IC50)is 0.317 ng/mL,the linear detection range(IC20-IC80)is 0.136-1.181 ng/mL,and the minimum detection limit(LOD,IC10)is 0.081 ng/mL.After optimization,the optimal pH of the detection system is 7.4,the salt concentration is≤0.4 mol/L,the content of methanol,ethanol,ace-tone and DMSO is≤5％,the content of acetonitrile is≤10％,and the concentration of bovine urine in the detection matrix is≤15％.The detection method has good stability and specificity.The addition recovery rate of intra-batch and inter-batch repeated test samples is 101％-1 12％and 101％-120％respectively,and the coefficient of variation is less than 10％,The correlation coefficient between the test results and HPLC-MS test results is R2=0.990 6.The detection method is fast,sensitive,accurate,reproducible and low detec-tion limit,and is suitable for high-throughput detection and on-site rapid screening of ZIL residues.

本试验旨在筛选和鉴定与猪程序性死亡因子1(PD-1)及其配体(PD-L1)相互作用的表位多肽,为阻断猪PD-1/PD-Ls通路逆转机体的免疫功能提供新策略.根据已解析人与鼠的PD-1与PD-L1相互作用的关键氨基酸位点信息,分析猪PD-1与PD-L1蛋白相互结合的关键氨基酸位点,在关键氨基酸位点处设计系列表位多肽,固相合成法合成表位多肽;分离自然感染猪圆环病毒2型(PCV2)仔猪的外周血单个核细胞(PBMC),检测表位多肽与猪重组蛋白PD-1、PD-L1和PBMC的结合能力,选取能结合猪PD-L1和PBMC的表位多肽pPD-15,检测其对猪PBMC增殖的影响,分析其作为佐剂免疫后对小鼠猪瘟病毒(CSFV)抗体水平的影响,最后,高效液质联用色谱法(HPLC-MS)检测表位多肽pPD-15的纯度和氨基酸序列正确性.结果显示,表位多肽pPD-15能结合猪PD-L1和PBMC;增殖试验显示,pPD-15组M1的平均荧光强度(74.20％)比空白对照组M1的平均荧光强度(4.37％)提高了 69.83％,表明表位多肽pPD-15可促进猪PBMC的增殖;动物免疫试验显示,pPD-15作为佐剂可以提高CSFV抗体水平;HPLC-MS结果显示,合成的表位多肽pPD-15纯度高,氨基酸序列正确.本试验证实,表位多肽pPD-15具有提高体内外免疫力的能力,为研制新型免疫调节佐剂提供了科学依据.

为建立一种检测猪圆环病毒3型(porcine circovirus type 3,PCV3)的PCR-ELSIA方法,根据GenBank上报道的PCV3全基因组序列,针对其高度保守区域设计1对特异性引物,上、下游引物的5'端分别标记生物素和地高辛.经PCR扩增获得大量带有标记的目的基因,依靠生物素-链霉亲和素的非共价结合作用固定目的基因于载体上,再通过羊抗DIG-HRP的显色反应,建立PCR-ELISA检测方法.结果表明,该方法与猪繁殖与呼吸综合征病毒、猪瘟病毒、猪细小病毒和猪圆环病毒2型无交叉反应,特异性良好.PCR-ELISA方法最低检测限为5.58×102 copies/μL,敏感性比常规PCR高100倍(常规PCR的检测限为5.58×104 copies/μL).PCR-ELISA方法批内和批间重复性检测的变异系数分别为1.90％～5.20％和3.89％～9.03％.对132份临床样品进行PCV3检测,PCR-ELSIA检出率为9.10％(12/132),高于常规PCR的检出率(4.55％,6/132).综上,该方法具有特异、灵敏、安全高效的优点,可为PCV3的诊断、防控和流行病学调查提供新的技术手段.

为制备齐帕特罗(ZIL)完全抗原,获得抗ZIL多克隆抗体(pAb),将ZIL与4-溴丁酸乙酯反应后,采用EDC/NHS方法使ZIL-丁酸盐中间体与牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)进行偶联,分别用紫外全波长扫描(UV)、聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定后,通过免疫新西兰大白兔获得抗ZIL多抗血清,并对抗体效价、半数抑制浓度(IC50)和特异性进行鉴定.HPLC-MS检测结果显示,改造后的主产物分子质量为348.21 u,约占整个反应体系产物的92.23％,与ZIL-丁酸盐分子质量完全相符;SDS-PAGE电泳结果显示,BSA/OVA在电泳中的迁移速率明显大于偶联产物;UV结果显示,相比载体蛋白,偶联产物ZIL-BSA/OVA的特征吸收峰均发生了明显变化;ELISA效价检测结果表明,ZIL多抗血清效价在1:6400以上,通过建立标准曲线测得IC50分别为0.38 ng/mL和1.13 ng/mL,并且与沙丁胺醇(SAL)、特布他林(TBL)、溴布特罗(BRO)、西马特罗(CIM)、克仑特罗(CLB)、盐酸多巴胺(DH)、莱克多巴胺(RAC)、达氟沙星(DAN)、泰乐菌素(TYL)均不存在任何交叉反应.成功制备了ZIL完全抗原ZIL-BSA/OVA,获得了一种高灵敏度和高特异性的抗ZIL pAb,为后续建立ZIL免疫学快速检测方法奠定了基础.

目的 建立一种以单链抗体为基础的酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)快速检测畜禽产品中西马特罗(cimaterol,CIM)药物残留.方法 以重氮化法合成的西马特罗完全抗原CIM-牛血清白蛋白(bovine albumin,BSA)免疫新西兰大白兔,用兔脾细胞总RNA反转录合成cDNA第一链,设计引物扩增兔源抗体重链可变区(heavy chain variable region,VH)和轻链可变区(light chain variable region,VL),通过45 bp的连接肽(Linker)使用新型重叠延伸聚合酶链式反应技术(splicing by overlap extension polymerase chain reaction,SOE-PCR)连接成VH-Linker-VL并大量制备;利用噬菌体表面展示技术将单链抗体(single-chain variable fragment,ScFv)片段与噬菌体展示载体pCANTAB-5e连接,电转化至大肠杆菌TG1感受态细胞,得到兔源噬菌体ScFv抗体库.通过聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)判断是否偶联成功,用紫外(ultraviolet,UV)扫描及基质辅助激光解析串联飞行时间质谱(matrix-assisted laser desorption tandem time-of-flight mass spectrometry,MALDI-TOF-MS)测定人工抗原的偶联比.用ELISA测定免疫兔血清质量.鉴定合成抗体库并对抗体库进行4轮淘选,取最优的CIM-ScFv进行氨基酸序列分析.结果 CIM-BSA及CIM-鸡卵清白蛋白(ovalbumin,OVA)偶联比分别为8:1及10:1,免疫血清校价为1:32000,半数抑制浓度为9.77 ng/mL,标准曲线的拟合度为0.997.抗西马特罗兔源噬菌体ScFv免疫库的插入率为92％,库容为1×1010 pfu,多样性为83.3％,经淘选获得一株与CIM有结合活性的噬菌体ScFv菌.结论 西马特罗完全抗原的合成效果良好,可用于建立西马特罗检测方法,兔源噬菌体抗体库为快速获得CIM单链抗体奠定基础,为西马特罗快速检测提供了新思路.

旨在了解和掌握新乡市某规模化猪场大肠杆菌的耐药情况及耐药基因类型,为猪细菌病及其耐药性研究奠定基础.本研究从该猪场采集样本,通过平板划线法进行大肠杆菌的分离纯化,采用K-B纸片法进行药敏试验,并对多种耐药性基因进行PCR检测.试验共分离鉴定出15株大肠杆菌.药敏试验结果表明,分离菌株对氨苄西林、万古霉素、复方新诺明、氯霉素、四环素、庆大霉素、左氧氟沙星等多种药物具有多重耐药性;PCR扩增结果显示,这些菌株携带有氯霉素类的floR基因(100％)、β-内酰胺类的blaTEM基因(80.0％)、四环素tetM的基因(26.7％)等耐药基因,可能是导致大肠杆菌对相应抗生素产生耐药的原因.综上所述,这些猪源分离菌株大多为多重耐药大肠杆菌.

以合成沙丁胺醇(SAL)完全抗原为基础获得针对SAL的多克隆抗体,建立检测SAL的间接竞争ELISA(ic-ELISA)检测方法,为进一步开发SAL快速检测试剂盒奠定基础.为此,分别用混合酸酐法和活化酯法制备SAL-BSA免疫原和SAL-OVA检测原,免疫新西兰大白兔获得SAL多抗血清,建立检测SAL ic-ELISA检测方法.结果显示,成功合成了 SAL-BSA和SAL-OVA,其偶联比分别为17 ∶ 1和6.4 ∶ 1;免疫后兔血清抗体效价为1 ∶ 102 400,所建立ic-ELISA的半数抑制浓度(IC50)为24.84 μg/L,最低检测限(IC10)为0.78 μg/L,线性范围IC20～IC80为 3.16μg/L～80.1 μg/L;精密度(CV)％＜10％,回收率在98％～110％之间.特异性鉴定结果显示,SAL多抗血清除与侧链上含有叔丁基官能团的结构类似物具有较强的交叉反应外,不存在与其他同类分子的交叉反应.以上结果表明,成功建立了检测SAL的ic-ELISA检测方法,为进一步开发SAL快速检测试剂盒奠定了基础.

实践教学是对学生的创新能力、实践技能、科技素质进行综合培养的重要教育模式.然而,生物技术专业实践教学仍然存在教学内容与时代脱节、不够系统全面、创造性思维培养不够等缺陷.我们需增强实践教学的系统性和创新性、阶段性提升教师素质及知识、有效运用多种教学手段等方法来进行实践教学改革,对当代大学生的基础知识结构、实验实践技能、创新创优意识等方面进行综合性培养.

科研实践是培养具有创新精神和创新能力人才的重要途径,也是新时期提升高校科研能力的重要方法.文章以新乡学院为例论述了对于本科生科研能力的培养中专业竞赛的重要性.较早的参加专业竞赛,是高校本科生科研能力得以加速提升的一种重要途径,尤其是普通本科院校或地方型院校,对于提高本科生创新精神和创新能力及高校科研能力具有极大的促进作用.

免疫系统中基因的精确表达和调控是生物体产生对病原体强大免疫能力及限制自身免疫的重要因素.越来越多的研究表明,长链非编码RNA(lncRNA)与免疫功能密切相关,能够通过调控免疫细胞分化发育以及免疫细胞效应功能参与免疫反应的调节.论文结合lncRNA调控作用的分子机制,阐述相关lncRNA在免疫应答和免疫调节过程中发挥的生物学效应,以期为从事lncRNA调控免疫应答机制研究提供参考.

生命科学导论是生物学相关专业大学一年级新生的一门重要专业基础课.然而在传统的教学体系中存在诸多问题,阻碍了学生综合能力的提高.教学实践中要不断探索,通过更新课程内容,强化"学生本位",构建网络资源,案例启发,合作专题,注重过程评价等一系列教学改革措施,有效培养了学生探究、创新、终身学习等能力.

文章旨在评估日粮添加不同水平的海藻对肉鸡生长性能、抗氧化及免疫性能的影响.试验将840只平均体重为(58.01±0.67)g的1日龄肉鸡随机分为4组,每组5个重复,每个重复42只鸡.对照组和处理组分别饲喂基础日粮+0、1.5％、2.5％和3.5％海藻,试验共开展42 d.结果:与对照组相比,3.5％海藻组42 d肉鸡体重显著提高1.39％(P<0.05),但22～42 d采食量及1～42 d料重比较对照组显著降低5.19％和4.80％(P<0.05).3.5％海藻组22～42 d肉鸡料重比较对照组和1.5％海藻组分别显著降低7.2％和5.69％(P<0.05).对照组血清胆固醇、甘油三酯和高密度脂蛋白浓度显著高于3.5％海藻组(P<0.05),同时,对照组和1.5％海藻组血清总脂浓度较3.5％海藻组分别显著提高11.86％和7.73％(P<0.05).与对照组相比,处理组血清超氧化物歧化酶活性分别显著提高10.78％、12.49％和11.89％(P<0.05).与对照组相比,1.5％～3.5％海藻组42 d肉鸡抗绵羊红细胞抗体滴度分别显著提高20.63％、15.61％和18.52％(P<0.05).结论:在本试验条件下,综合考虑生长性能、血清抗氧化和抗体滴度,1～42 d肉鸡日粮中海藻适宜的添加水平为3.5％.

长链非编码RNA(Long non-coding RNA,lncRNA)是缺乏开放阅读框架,且长度大于200 nt不编码蛋白质的转录本,其绝对数量大、种类多,在表达模式上具有明显的细胞特异性.lncRNA通过碱基配对与DNA或RNA,或通过RNA高级结构与蛋白质结合,发挥多种生物学功能,共同构成了一个复杂而精细的分子调控网络.结合近年来对lncRNA的研究发现,在阐述lncRNA分子作用机制研究进展的基础上,简要阐述了lncRNA在动物生产性能及动物生长发育方面的相关研究,旨在为进一步研究lncRNA的生物学调节功能提供参考,为lncRNA在畜牧业方面的应用提供科学依据.

程序性死亡因子-1(PD-1)作为调节或平衡机体外周和中枢免疫功能的重要分子,其在不同的抗原刺激、炎症环境、细胞种类及分化阶段的表达受复杂、多样的分子机制调控.为深入理解PD-1表达调控的分子机制,本文分别从顺式作用元件、转录调控因子及相关信号通路、表观遗传因素、转录后调控等几个方面,将已有关于PD-1表达调控分子机制的相关研究进展进行综述,以期为更精确、有效的免疫检查点疗法或治疗方案的设计提供新的信息和思路.