Fowl adenovirus serotype 11 (FAdV-11) is an emerging pathogen associated with inclusion body hepatitis (IBH) and high mortality in commercial broilers. In this study, we evaluated the protective efficacy of maternal antibody transfer against FAdV-11 in neonatal chicks. A hypervirulent FAdV-11 strain (FAdV-11/HB2022/1), isolated from an IBH outbreak in Hubei Province, China, caused 90% mortality in 1-day-old specific-pathogen-free (SPF) chickens; 7-day-old chicks exhibited no mortality but developed mild hepatic lesions and sustained viral replication. A soluble, trimeric Fiber subunit vaccine (Fiber282–569) was developed through rational truncation of the transmembrane domain, retaining both the shaft and head domains, with its homotrimeric conformation confirmed by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). Immunization of breeder hens with Fiber282–569 elicited high titers of FAdV-11-specific IgY and neutralizing antibodies that were efficiently transferred to progeny via the egg yolk. Upon lethal challenge with FAdV-11/HB2022/1, 1-day-old progeny chicks from vaccinated breeders exhibited 100% survival with no clinical signs, no gross pathological lesions, and undetectable viral loads in liver tissues, demonstrating complete protection. In contrast, all chicks from unvaccinated breeders succumbed within 5 days post-infection. These results demonstrate that maternal antibody transfer of the fiber-based subunit vaccine confers complete protection against lethal FAdV-11 infection in neonatal chicks, providing a safe and effective candidate strategy for controlling FAdV-11 in the poultry industry.

The growing prevalence of hyperuricemia necessitates the urgent development of more potent treatments. This study aimed to develop, optimize, and evaluate the safety and efficacy of porcine-human recombinant uricase (PHRU) both in vitro and in vivo. The study employed gene editing of PHRU through site-directed mutagenesis, with recombinant proteins expressed in vitro utilizing Escherichia coli. The polyethylene glycol (PEG) approach was employed to augment uricase stability and diminish immunogenicity. The pharmacokinetics and pharmacodynamics of PHRU were tested in vitro and in Sprague Dawley rats. Successful expression of the fusion protein in E. coli and the development of the PEGylated drug were achieved. In vitro experiments confirmed the efficacy of PEG-PHRU in degrading uric acid, with PEGylation not markedly affecting the biological activity of PHRU. Animal studies revealed that PEG-PHRU significantly lowered plasma uric acid levels and mitigated hyperuricemia-induced renal damage in rats. Both drug metabolism and pharmacokinetics exhibited favorable characteristics without observable adverse effects in experimental animals. This novel fusion protein shows the potential for ameliorating hyperuricemia and related renal complications, highlighting it as a promising drug candidate with substantial market applications.

Recombinant human acidic fibroblast growth factor (rh-aFGF) is a widely used biological product, but it is unstable and its biological activity is easy to decrease. In order to maintain the long-term stability and biological activity of rh-aFGF, based on the response surface method, the freeze-drying characterization and cell proliferation rate of rh-aFGF freeze-dried powder were evaluated by scoring and Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay in this study. The optimal concentrations of trehalose, glycine and BSA were optimized, and the optimal formulation was verified by regression experiment. The results showed that trehalose, glycine and BSA had significant effects on the characterization of lyophilized rh-aFGF and cell proliferation. The optimal formulation of 5.7% trehalose, 2.04% glycine and 1.98%BSA combined with rh-aFGF could achieve the optimal freeze-dried characterization and biological activity. Using the best formulation to verify, the freeze-dried formability index of the freeze-dried powder was 23.35, and the rate of cell proliferation was 43.59%, which was close to the expected 23 and 41.69%. This study determined a freeze-dried formulation of rh-aFGF that meets the requirements of freeze-dried formalization integrity and maintains biological activity, providing reliable support for the subsequent development of related drugs.

禽腺病毒是全球家禽常见的传染病病原之一,目前对禽腺病毒进行流行病学调查,已给全球家禽业造成了巨大的经济损失.为了调查禽腺病毒在湖北地区的流行情况,在2021～2022年从湖北市场收集了224份鸡肝样本,为FAdV疫苗研制提供参考.基因测序结果显示:其中14株携带FAdV,Hexon L1环基因序列系统发育,12株为C型,2株为D型.在鸡肝癌细胞株(LMH)中培养并分离得到FAdV-11 HB200株,对分离物的Hexon基因进行了扩增和测序.遗传进化分析表明:FAdV-11 HB200与多个FAdV-11毒株处于同一分支.FAdV-C的流行率仍然占主导地位,相关部门应采取预防FAdV感染的措施.

Superoxide dismutase (SOD) is an antioxidant enzyme with multiple metal cofactors that can specifically clear reactive oxygen species (ROS), which plays an important role in a variety of ultraviolet-induced lesions. Therefore, SOD has the anti-ultraviolet radiation effect. The objective of this study was to compare the differences in the anti-ultraviolet radiation effect of SOD with distinct metal cofactors: Cu/Zn-SOD and Mn-SOD. SOD was first purified using hydrophobic interaction chromatography and ion-exchange chromatography. Second, the Methylthiazolyldiphenyl-tetrazolium bromide method and cell senescence kits were used to study the protective effect of SOD on ultraviolet-induced cell damage. Finally, the protective effect of SOD on ultraviolet -induced skin damage was histopathologically evaluated, and the expression levels of malondialdehyde (MDA) and matrix metalloproteinases (MMPs) in tissues were detected. The results showed that Cu/Zn-SOD was superior to Mn-SOD in promoting cell proliferation, alleviating cell damage, protecting skin structure, and regulating the expression levels of MDA and MMPs, and it has no side effects. In conclusion, Cu/Zn-SOD had a better anti-ultraviolet radiation effect than Mn-SOD, and it can be used in anti-aging and anti-ultraviolet skin-care products.

为了调查鸡传染性贫血病病毒(CAV)在肉鸡群中的流行情况及CAV毒株的致病性,于2021年在湖北荆州地区收集鸡肝样品224份,采用PCR方法进行检测,并对CAV的全基因扩增片段克隆测序,用生物信息学软件对测序结果进行遗传进化及分子特征分析;通过动物回归试验探究分离毒株JZ2118的致病性.结果显示,18份样品为CAV阳性,CAV阳性率为8.04％.CAV全基因序列与CAV VP1氨基酸序列的遗传进化树分组基本相同,说明CAV的遗传进化主要与VP1蛋白氨基酸的突变有关;1日龄SPF鸡感染JZ2118毒株14 d后致死率高达82.6％,在体重、病毒载量、血常规检测与病理组织切片等方面都具有显著性变化,表明JZ2118分离株是具有较强致病性的强毒株.

制备禽腺病毒4型(FAdV-4)Fiber2蛋白的特异性单克隆抗体,将重组蛋白FAdV-4 Fiber2纯化后作为免疫原免疫BALB/c小鼠,分离脾脏中的淋巴细胞,与小鼠骨髓瘤细胞SP2/0进行融合,筛选得到10株能稳定分泌抗FAdV-4 Fiber2蛋白单克隆抗体的阳性杂交瘤细胞株1B5F1、1B5G11、1B5C1、1B5D2、1B5E3、1B5F3、1B5E4、1B5G4、1B5A8、1B5G8,取分泌抗体水平最高的1B5F1细胞株制备腹水,利用斑点杂交(Dot blot)和间接免疫荧光试验(IFA)进行特异性检测.结果表明,成功制备10株杂交瘤细胞株;制备的腹水能与FAdV-4 Fiber2蛋白及FAdV-4攻毒后的鸡肝组织产生特异性反应,证明成功制备了FAdV-4 Fiber2蛋白特异性单克隆抗体.

对河南某兔场出现呼吸困难和腹泻等症状的2月龄兔群进行临床诊断及实验室检测.采用临床问诊、病理剖检、病原菌分离培养、染色镜检、PCR鉴定及药物敏感试验等方法,确定病原菌种类,筛选敏感药物,为发病兔场提供科学合理治疗方案.结果在发病兔体内分离到多杀性巴氏杆菌(Pasteurella multocida,PM)和肺炎克雷伯氏菌(Klebsiella pneumoniae,KP)2种病原菌,且2种菌均对庆大霉素、新霉素、四环素、多西环素、多黏菌素B、丁胺卡那6种药物敏感,对米诺环素、复方新诺明、恩诺沙星、杆菌肽4种药物不敏感.粪便球虫卵囊检测结果显示:其每克粪便卵囊数量在正常范围内.通过临床诊断及PCR结果综合判断,该兔场发病是由于PM和KP混合感染引起.根据药敏试验结果,建议兔场采用新霉素和多西环素联合用药,兔群在用药后第3天腹泻症状得到有效控制,1周后兔群基本恢复正常.

为了 分析猪圆环病毒2b 型病毒样颗粒(virus-like particles of Porcine circovirus type 2b,PCV-2b VLPs)表面具有外源肽展示潜力的位点,试验通过PyMol软件分析了 PCV-2b VLPs表面的位点分布,并以高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)Gp5的中和表位B(Epitope B,EpB)为外源肽,通过重叠PCR方法将表面位点替换为EpB,构建EpB嵌合的PCV-2b衣壳蛋白(Cap),利用原核表达系统诱导表达后进行凝胶层析纯化和Western-blot检测,并利用透射电子显微镜分析VLPs的组装状态.结果表明:在PCV-2b VLPs表面共筛选到8个可用于外源肽展示的位点,分别为S1(Y50TIKRTTVKTP60)、S2(L75PPGGGSN82)、S3(F124VTKATAL131)、S4(V161LDSTIDY168)、S5(Q183TAGNVD189)、S6(1201 YDQE205)、S7(K222DPPL226)、S8(S104PITQGDRGVGS115)位点;将EpB 替换展示到S1～S8位点后,构建的EpB嵌合的PCV-2b Cap能够在原核表达系统中获得不同程度的可溶性表达,其中S1～S7位点被EpB替换后能够实现较高水平的可溶性表达;8个目的蛋白均能被抗EpB抗体特异性识别,也均能够自组装形成VLPs,但粒径大小和形态与PCV-2b VLPs有不同程度的差异.说明试验筛选出的8个位点均有一定的外源肽展示能力.

Hepatitis-hydropericardium syndrome (HHS) is widespread in China and causes high chicken mortality that results in great economic losses. A safe and effective vaccine is needed, and a subunit vaccine has potential for development. In this study, a truncated region of the FAdV-4 fiber 2 fused with coding sequence of one epitope of hexon was expressed in a prokaryotic expression system, and the immune protective effects of different doses of recombinant fiber 2 subunit vaccine on SPF chickens were compared. The recombinant fiber2 (Gly275- Pro479 aa)-hexon (Met21-Val51 aa) protein (rFH) obtained in Escherichia coli showed good solubility. The chicken survival rate at the lowest dose (2.5 mu g/bird) was 75% (6/8), and at higher doses (>5 mu g/bird) was 100% (8/8) in challenge experiment. Two chickens in the 2.5 mu g/bird treatment showed severe lesions, while birds in the higher dose treatments showed no obvious tissue damage as determined by histopathologic analysis of liver and spleen. Absolute quantitative real-time PCR showed no viral load in the >5 mu g/bird treatments, but two chickens in the 2.5 mu g/bird treatment had high viral loads. The challenge experience demonstrated that the rFH vaccine provided 100% protection at >5 mu g/bird. These results suggested that rFH protein as an effective vaccine to protect against FAdV-4 and provided a new idea for the development of vaccine against HHS.

目的 对重组猪尿酸氧化酶(recombinant porcine urate oxidase,rPUOX)进行突变改造,以增加rPUOX上可供聚乙二醇(polyethylene glycol,PEG)耦合的赖氨酸(Lys)残基数,探究PEG修饰对改造前后的酶液在SPF鸡体内的药效和抗原屏蔽作用的影响.方法 对纯化的基因改造前后的rPUOX进行PEG化修饰,修饰蛋白静脉注射SPF鸡,共4次,每次间隔7d.实验分为20×5K PEG-rPUOX-215组、20×5K PEG-rPUOX组和空白对照组(0.9％生理盐水).于鸡翼根静脉处采血,用血糖尿酸测试仪及尿酸测试条测定尿酸浓度,酶反应-紫外分光光度法检测尿酸酶活性,间接ELISA法检测免疫原性.结果 20×5K PEG-rPUOX-215和20×5K PEG-rPUOX均获得充分修饰,且前者修饰效果更好.第1、2次注射,降低鸡血尿酸幅度20×5K PEG-rPUOX-215组高于20×5K PEG-rPUOX组约11％和6％.3次注射药效维持时间20×5K PEG-rPUOX组相较于20×5K PEG-rPUOX-215组逐次降低0、24和48 h.20×5K PEG-rPUOX-215组抗体产生速率相对较慢,且14 d抗体阳性率低于20×5K PEG-rPUOX组约14.3％.结论 20×5K PEG-rPUOX-215修饰后药效学作用更显著,药物半衰期延长,且免疫原性相对较低,为研制低免疫原性的rPUOX及实现临床上多次给药提供了新的方法和思路.

为建立一种检测非洲猪瘟病毒(ASFV)抗体的间接ELISA(iELISA)方法,对构建的ASFV P30基因表达工程菌诱导表达后,将获取的重组ASFV P30蛋白进行纯化和Western-blot检测,然后以纯化的重组蛋白为抗原,建立了ASFV抗体iELISA检测方法,并进行了特异性、灵敏性、重复性试验;同时与基于ASFV P30-2His6蛋白(N、C末端各融合1个His6标签)的iELISA方法进行兽医临床样本比较试验.结果显示:重组ASFV P30蛋白和重组ASFV P30-2His6蛋白在Western-blot检测中,均能与猪ASFV阳性血清产生特异性杂交带;基于重组ASFV P30蛋白iELISA的最佳反应条件为,抗原蛋白包被质量浓度20μg/mL、血清样品稀释度1:1000、酶标二抗稀释度1:40000、血清样品检测OD450阳性结果临界值0.22.该方法仅对ASFV阳性血清呈特异性反应,1:3200稀释的阳性血清仍可检出,批内试验和批间试验变异系数均小于10％,可以消除His标签所造成的假阳性反应.本研究建立的ASFV P30 iELISA检测方法为ASFV抗体检测提供了一种有效手段.

The Cap of porcine circovirus type 2 (PCV2) can be assembled into virus like particles (VLPs) in vitro that have multiple loops located on the particle surface. This would make it a good vehicle for displaying exogenous proteins or epitopes. We derived two epitopes, epitope B (EpB, S37HIQLIYNL45) and epitope 7 (Ep7, Q196WGRL200) from Gp5 of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). We replaced the core region of Loop CD (L75PPGGGSN82) and the carboxyl terminus (K222DPPL226) of PCV2 Cap, respectively, to construct a bi-epitope chimeric PCV2 Cap. Its immunogenicity and protective effects were evaluated as one PRRSV subunit vaccine. The chimeric PCV2 Cap was soluble, efficiently expressed in an Escherichia coli expression system, and could be self-assembled into chimeric virus like particles (cVLPs) with a diameter of 12-15 nm. Western blotting confirmed that the cVLPs could be specifically recognized by anti-PCV2, anti-EpB and anti-Ep7 antibodies. The cVLPs vaccine could alleviate the clinical symptoms and reduce the viral loads after HP-PRRSV challenge in 100-120 days old pigs. These data suggest that the cVLPs vaccine could provide pigs with partial protection against homologous PRRSV strains, and it provides a new design for additional PRRSV subunit vaccines.

猪圆环病毒2型(porcine circovirus type 2,PCV2)是一种广泛存在于猪体内的DNA病毒.养猪场中猪的PCV2感染率较高,为区分自然感染猪和疫苗免疫猪,以大肠杆菌表达的PCV2 Rep蛋白为包被抗原,通过优化反应条件建立PCV2 Rep抗体检测的间接ELISA方法.原核表达的Rep蛋白大小40ku,经过变性亲和层析纯化后目的蛋白纯度为95.1％,批次内和批次间血清检测变异系数均低于10％,表明Rep-ELISA具备良好的稳定性.血清学交叉试验显示Rep-ELISA具有良好的特异性;猪免疫试验结果表明Rep-ELISA能检测出PCV2全病毒疫苗免疫以及接种PCV2 SD/2017活毒的猪血清中的Rep抗体,而不能与PCV2亚单位疫苗免疫猪血清中的抗体发生免疫结合反应.研究结果表明,所建立的ELISA方法能有效鉴别自然感染或全病毒灭活疫苗免疫猪与亚单位疫苗免疫猪,可用于猪群中PCV2的净化.

Introduction. PCV2 is a DNA virus that exists widely in pigs and has caused great economic losses to the pig industry worldwide. In the existing commercial PCV2 enzyme-linked immunosorbent assay (ELISA) kits both natural infection with PCV2 and vaccine immunization produce results that are positive for PCV2 Cap antibodies and therefore they cannot diagnose PCV2 infection in immunized pig farms.Aim. To establish a PCV2 non-structural protein antibody detection method that distinguishes between antibodies resulting from natural prior exposure (infection) and those induced by subunit vaccine immunization.Methodology. Based on the non-structural Rep' protein, we established an indirect ELISA (iELISA) using sera from guinea pigs and piglets.Results. The results for iELISA for guinea pig serum showed that animals vaccinated with a whole-virus inactivated PCV2 vaccine had 100 % (10/10) Cap antibody positivity and 100 % (10/10) Rep' antibody positivity. Guinea pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (10/10) Cap antibody positivity, while no (0/10) guinea pigs were Rep' antibody-positive. The combined detection results for the Rep' iELISA and a PCV2 Antibody Test kit (Commercial) showed that pigs vaccinated with a whole-virus inactivated PCV2 vaccine or PCV2 SD/2017 had 100 % (5/5) Cap antibody positivity and 100 % (5/5) Rep' antibody positivity. Pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (5/5) Cap antibody positivity, while no (0/10) pigs were Rep' antibody-positive.Conclusion. This paper describes an effective iELISA method that can distinguish natural infection with PCV2 (Cap and Rep positive) or inoculation with a whole-virus inactivated vaccine (Cap and Rep positive) from subunit vaccine immunization (Cap-positive, Rep-negative). These comparative assays could be very useful in the control of PCV2 in pig herds.

Since 2017, novel variant strains of infectious bursal disease virus (nvIBDV) have been detected in China, while the current vaccines on the market against very virulent IBDV have limited protection against this subtype virus. In this context, a strain of the virus has been isolated, and sequencing alignment and bird regression experiments showed that the virus was IBDV, belonging to the nvIBDV subtype (and named IBDV FJ-1812). Furthermore, the Escherichia coli expression system was used to successfully express soluble nvIBDV rVP2, which is specifically recognized by an anti-IBDV standard serum and anti-nvIBDV positive serum, and could be assembled into 14 - 17 nm virus-like particles. Based on the purified nvIBDV rVP2, we developed an IBDV FJ-1812 VP2 VLP vaccine at a laboratory scale to evaluate protection by this vaccine; in addition, we also prepared an IBDV JZ 3/02 VP2 subunit vaccine targeting very virulent IBDV and evaluated its cross-protection against nvIBDV. Results of bird experiments showed that the nvIBDV rVP2 vaccine could induce high titres of specific antibodies, completely protect the bursa of Fabricius from viral infection, and provide 100% immune protection to SPF and Ross 308 broiler chickens. Furthermore, the IBDV JZ 3/02 VP2 subunit vaccine targeting very virulent IBDV could provide 60% protection for SPF chickens and 80% protection for Ross 308 broiler chickens. This report provides important technical supports for the prevention and control of nvIBDV in the future.

为获得较高纯度的猪圆环病毒2型(porcine circovirus type 2,PCV2) Cap蛋白的病毒样颗粒(virus-like particles,VLPs)并对其进行结构和功能研究,本试验以大肠杆菌表达系统表达了PCV2重组Cap (PCV2 rCap)蛋白,通过硫酸铵分级沉淀、Sephacryl S-300 HR凝胶过滤层析、Capto Q离子交换层析、CsCl密度梯度离心等方法对其进行了纯化.通过Western blotting对纯化所得蛋白进行免疫反应性鉴定,透射电镜(TEM)观察纯化蛋白的粒径及形态,高效液相尺寸排阻色谱(HPSEC)检测其组分分布.结果 显示,纯化后PCV2 rCap蛋白经SDS-PAGE检测,可见28 ku的目的蛋白带,灰度扫描法检测其纯度为97.53％.Western blotting结果显示,该处的特异性蛋白条带有明显的免疫反应性;TEM检测其为粒径17.35～19.24 nm的规则球形颗粒,表明所获得的PCV2 rCap蛋白在表达时能在菌体内自我装配成VLPs,且在纯化过程中没有被破坏而解聚,仍保持天然状态;HPSEC检测纯化样品中VILPs含量为92.67％.本试验结果为进一步研究PCV2 VLPs的空间结构及其免疫保护机制提供了参考依据.

为了探讨猪繁殖与呼吸综合征病毒(PRRSV)N蛋白基因工程亚单位疫苗的免疫保护效果,试验利用大肠杆菌表达并纯化了重组PRRSV N蛋白,添加佐剂制成亚单位疫苗进行免疫保护试验.试验猪分为2个不同佐剂制备的PRRSV N蛋白亚单位疫苗免疫组,1个PRRSV TJM-F92株减毒活疫苗免疫组和1个攻毒对照组.首免5周后,用PRRSV NVDC-JXA1强毒株攻击,观察临床表现并在攻毒后21 d对试验猪进行剖检.结果表明:首免3周后2个亚单位疫苗组和减毒活疫苗组抗体全部转阳,组间无显著性差异(P＞0.05);攻毒后PRRSV N蛋白亚单位疫苗免疫1#组有2/3的猪只、2#组有1/3的猪只的临床症状、体征和解剖病变较攻毒对照组猪只轻微,转归较好且成活,但保护作用不及PRRSV TJM-F92株减毒活疫苗组;剩余的亚单位疫苗免疫1#组1/3的猪只和亚单位疫苗免疫2#组2/3的猪只,与攻毒对照组猪只一样表现为比较典型的PRRS症状,并在8～12d内死亡.说明PRRSV N蛋白亚单位疫苗能够引起机体的体液免疫反应,具有部分保护作用,但不能提供完全的保护,单独使用达不到减毒活疫苗的效果.

为建立以豚鼠为模型动物检测猪圆环病毒2型(PCV2)疫苗效果的方法,本研究以Hartley豚鼠为模型动物,采用原核细胞表达的PCV2 Cap蛋白作为包被抗原,建立了检测PCV2抗体的间接ELISA方法,进行PCV2疫苗免疫效果的评价.结果表明,该方法检测未免疫豚鼠血清的OD450nm本底值较低,平均值为0.0673.依据138只未免疫豚鼠的OD450nm平均值确定ELISA临界值:S/P值≥0.2为阳性;S/P＜0.1为阴性;0.2＞S/P≥0.1为可疑.该检测方法具有很高的特异性和较好的可重复性.采用国内和国外两种PCV2疫苗免疫后,豚鼠血清抗体滴度在4周内达到峰值,两组之间变化趋势高度相关,Pearson相关系数为0.805.该检测方法能够敏感的显示被免疫豚鼠血清抗体从阴性变为阳性的过程.5种不同生产商的PCV2疫苗免疫后测定的S/P比值能够较好的反映血清抗体的变化,各组豚鼠的S/P平均值分别为:0.1645、1.1875、0.5166、1.3559及3.0907.各组的抗体阳性率分别为:40％、40％、20％、100％及100％.以豚鼠为模型动物的间接ELISA方法是一种理想的PCV2疫苗免疫效果评价方法,为疫苗生产厂商PCV2疫苗的开发和养殖企业的疫苗筛选提供了可靠而实用的技术.