Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), is an acute and highly contagious intestinal disease that inflicts substantial economic losses on the global swine industry. The nucleocapsid (N) protein of PEDV plays a critical role during viral infection and replication. In this study, the full-length N gene was cloned and expressed using the prokaryotic expression vector pET-32a (+). The purified recombinant N protein was used to immunize BALB/c mice. Subsequently, splenocytes from the immunized mice were fused with SP2/0 cells, and hybridoma cell lines secreting monoclonal antibodies (mAbs) against N protein were screened via indirect ELISA. The linear B-cell epitopes recognized by the mAbs were mapped using truncated N protein fragments. Results showed that three stable hybridoma cell lines (1A3, 1G1 and 1A10) secreting N protein-specific mAbs were obtained. Epitope mapping revealed that mAbs 1A3 and 1G1 recognized the epitope 71SNWHF75, whereas mAb 1A10 recognized 66RIEQP70. Bioinformatics analysis indicated that these epitopes are highly conserved among the analyzed PEDV strains and show no cross-reactivity with the N proteins of other coronaviruses. These findings could provide valuable experimental materials for further investigation of the N protein’s structure and function and support the development of diagnostic assays and subunit antigen vaccine for PEDV.

Dendritic cells (DCs) bridge innate sensing and adaptive immunity. Their functional heterogeneity derives from fundamental differences among DC subsets in ontogeny, tissue distribution, receptor repertoires, and antigen-processing pathways, establishing a specialized “division of labor” in antigen presentation, encompassing classical major histocompatibility complex (MHC) II-restricted presentation of exogenous antigens, MHC I-restricted presentation of endogenous antigens, and differences in the efficiency and preference of antigen cross-presentation. These subset-intrinsic features enable DCs to selectively instruct naïve T cells toward cytotoxic immunity, helper lineage differentiation, or immune tolerance. Recent advances reveal previously unappreciated dimensions of DC specialization. Type 1 conventional dendritic cells (cDC1s) not only excel in cross-presentation but also shape tumor immunogenicity through CLEC9A (also known as DNGR-1)-mediated recognition of F-actin-associated neoantigens, driving immune editing. Type 2 conventional dendritic cells (cDC2s) orchestrate lymph node dynamics-recruiting innate effectors to form transient “immune firewalls” and subsequently restoring tissue architecture for efficient T cell priming. In this review, we delineate how subsets-specific differences dictate specialized functions and shape immune outcomes. This review discusses DC subset biology from the perspective of veterinary vaccine research, with attention to both conserved mechanisms and species differences. We also summarize how DC-targeted delivery may be considered in veterinary precision vaccinology. Insights from large-animal models offer unique opportunities to validate next-generation vaccines against major animal diseases while informing human vaccine design through bidirectional cross-species translation.

The Complementarity-determining region (CDR) is the core region that determines the functions and characteristics of nanobodies. To analyze the impact of the CDR3 region on the developability of nanobodies and antigen-binding activity, this study used two porcine CD205-specific nanobodies (Nb205-1 and Nb205-10) with different CDR3 regions as models and compared them in terms of prokaryotic expression, Ni column purification, and antigen binding, providing references for the screening and evaluation of nanobodies. Phage display technology was employed to screen porcine CD205-specific nanobodies. Positive clones were subjected to sequence alignment, and Swiss-Model was utilized for homology modeling and structural alignment. The Escherichia coli system was employed to evaluate the strain proliferation capacity, in vitro protein expression form, and Ni-NTA affinity purification efficiency. ELISA was used to detect their binding activity with the target protein of porcine CD205 and their antigenic epitopes. Phage-ELISA results demonstrated that two nanobodies, Nb205-1 and Nb205-10, were screened out with high binding activity to the target protein of porcine CD205. Sequence alignment analysis revealed that the framework regions of the two nanobodies were highly conserved, with only several amino acid residue differences in the CDR3 region. Strain Nb205-1 exhibited superior developability, with high protein expression in E. coli, strong binding affinity to Ni-NTA resin, and ease of purification. In contrast, Nb205-10 existed in the inclusion body form, showed weak binding affinity to Ni-NTA resin, and had extremely low purification efficiency. The 3D structural modeling and electrostatic potential analysis revealed that the CDR3 loop of Nb205-1 presented a convex conformation and was rich in positive charges, while Nb205-10 exhibited a relatively flat concave conformation and neutral charge, suggesting that the two may recognize similar antigenic epitopes through different molecular mechanisms. Epitope analysis indicated that the antigenic epitope recognized by Nb205-1 was located in the region 1MITPLNDWIVAKDCDKTK18. This study identified two porcine CD205 nanobodies (Nb205-1 and Nb205-10) with similar binding activity, while their developability (expression and purification efficiency) was significantly influenced by sequence differences in the CDR3 region. In future therapeutic nanobody screening, comprehensive evaluation of their developability should be conducted to provide theoretical and practical bases for rational design.

Recently, CRISPR/Cas13a-based biosensors have been combined with non-traditional lateral flow assays (NTLFAs) successfully used to clinical detection of target RNA due to their simplicity, portability, and costeffectiveness. However, N-TLFAs used in such biosensors have several disadvantages, such as inversion of test and control zones, in which color intensity depends on reporter probe cleavage. Herein, we propose a novel biosensor in which CRISPR/Cas13a is integrated with traditional lateral flow assays (TLFAs) to facilitate the sequential direct location of test/control zones by dual hairpin probes, namely DNA hairpin reporter probe B-HPs (5 ' Biotin-hairpins, B-HPs, loop containing 5 rU) and F-HPs (5 ' FAM-hairpins, F-HPs). Cas13a activated by crRNAtargeted RNA duplexes can indiscriminately cleave B-HPs reporter probes to release short sequences to open hairpin F-HPs reporter probes to form Biotin-dsDNA-FAM reporter probes. The advantage of this platform is that detection results only rely on the formed Biotin-dsDNA-FAM reporter probes, which can be used for SARS-CoV-2 RNA detection after preliminary detection. Under optimized conditions, the SARS-CoV-2 RNA detection range was 10 aM to 1 mu M and the limit of detection was as low as 5.34 aM. This proof-of-concept study demonstrates that dual-hairpin reporter-probe binding to TLFAs opens up new opportunities for CRISPR/Cas13a activity detection and bioanalytical applications.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a pivotal role in bridging innate and adaptive immunity, making them a central focus in vaccine development. As a C-type lectin receptor expressed on cDC1 and cDC2 subsets, CD205 facilitates receptor-mediated endocytosis, enabling antigen presentation through both MHC class I and class II pathways, which are critical for activating cytotoxic and helper T cells. In this study, we introduced a CD205-targeted bispecific nanobody (BiNb-CD205/FMDV) as a novel platform for enhancing antigen delivery and immune activation of foot-and-mouth disease virus (FMDV) in pigs. In vitro experiments demonstrated that BiNb-CD205/FMDV could bind efficiently to porcine bone marrow-derived dendritic cells (BMDCs) and show a strong colocalization with acidic organelles such as lysosome, indicating significantly enhancing antigen uptake and effective processing. In vivo immunization results revealed Nb4-Nb205 was effective at enhancing LPB-specific antibody titers, inducing enhanced CD4+ and CD8+ T cell responses. Elevated cytokine levels, including IFN-γ and IL-4 further supported robust immune activation, indicating a balanced Th1/Th2 response. Our results provide preliminary evidence for the feasibility of CD205-targeted bispecific nanobody platforms in enhancing antigen presentation and immune responses. This highlights the potential to expand targeted delivery to the field of animal epidemic diseases and provides a reference for the general application of nanotechnology in viral diseases.

Avian infectious bronchitis is caused by the avian infectious bronchitis virus (IBV), which poses a significant threat to the poultry industry and public health. The S1 protein of IBV plays a crucial role in the process of the virus invading host cells. To investigate the significant antigenic targets within the S1 protein, in this study, the truncated S1 sequence of the IBV M41 strain was cloned with approximately 660 bp and expressed. After purification and renaturation, the recombinant S1 protein was immunized into BALB/c mice. Then, following fusion with lymphocytes and SP2/0 cells, the indirect ELISA and Western blotting techniques were employed to screen hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the S1 protein. Antigenic epitopes of the mAbs were identified using truncated S1 fragments and peptide scanning. The results indicated that three hybridoma cell lines stably secreting S1 protein-specific mAbs (2A10, 4E9, and 5E12) were screened. The heavy chains of the three mAbs were IgG1, and all three mAbs contained kappa light chains. The identified minimal B-cell epitopes were 132RVSAMK137 and 142FYNLTV147. Homology analysis showed these both epitopes were conserved across IBV subtypes and located on the S1 protein surface. The conserved β-sheet epitope 132RVSAMK137 and the surface-exposed, flexible loop epitope 142FYNLTV147 serve as ideal targets for broad-spectrum diagnostics and early infection detection, respectively. These epitopes provide unique structural advantages for antibody binding, enabling the design of multivalent epitope vaccines or the development of immunomodulatory drugs. They offer novel biomaterials and targets for antibody-based drug development and rapid detection methods for avian infectious bronchitis virus (IBV), holding significant potential for the prevention and control of IBV.

Classical swine fever virus (CSFV) infection induces complete mitophagy, which is essential for the clearance of damaged mitochondria. The endosomal sorting complex required for transport (ESCRT) machinery plays a vital role in mediating phagophore closure and autophagosome-lysosome fusion during starvation-induced autophagy. Nevertheless, its involvement in CSFV-induced mitophagy and the underlying mechanisms remain insufficiently understood. Here, we found that the ESCRT-III subunits including CHMP1A, CHMP1B, and CHMP4B, along with the AAA-ATPase VPS4, were actively recruited to autophagosomes during CSFV-induced mitophagy. Consistent with this, depletion of CHMP1A, CHMP1B, CHMP4B or VPS4A disrupted mitophagic flux, impairing both PINK1-PRKN-dependent and -independent pathways. Further investigations revealed that CSFV transiently recruited these subunits to nascent autophagosomes for phagophore sealing during mitophagy. Remarkably, multiple CSFV nonstructural proteins (NSPs) including NS3, NS4B, NS5A and NS5B interacted with these ESCRT key subunits and colocalized on mitophagosomes. Taken together, our study identifies CHMP1A, CHMP1B, CHMP4B, and VPS4A as pivotal regulators of phagophore closure in CSFV-induced mitophagy, unveiling novel mechanisms by which the virus manipulates host cellular pathways and highlighting potential therapeutic targets for infection control.Abbreviation: ATF4: activating transcription factor 4; ATG5: autophagy related 5; BafA1: bafilomycin A1; BFP: blue fluorescent protein; BNIP3L/NIX: BCL2 interacting protein 3like; BSA: bovine serum albumin; CALCOCO2/NDP52: calcium binding andcoiled-coil domain 2; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CHMP: charged multivesicular body protein; COX4: cytochrome c oxidase subunit 4; CSFV: classical swine fever virus; DAPI: 4',6-diamidino-2-phenylindole; DN: dominant-negative; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; hpt: hours post-transfection; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; IB: immunoblotting; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFF: mitochondrial fission factor; MFN2: mitofusin 2; MITO: mitochondria; MOI: multiplicity of infection; mtDNA: mitochondrial DNA; OPTN: optineurin; PBS: phosphate-buffered saline; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAPA: rapamycin; RFP: redfluorescent protein; RT-qPCR: reverse transcription-quantitativereal-time polymerase chain reaction; RT-PCR: real-time polymerasechain reaction; SD: standard deviation; siCtrl: negative control siRNA; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC1: voltage dependent anion channel 1; VPS4A: vacuolar protein sorting 4 homolog A; WCL: whole-cell lysate; WT: wild-type.

RESEARCH HIGHLIGHTS:The host protein CCT8 interacts with the influenza virus protein PB2.The CCT8 gene is upregulated during influenza virus infection.Knockdown of CCT8 inhibits viral proliferation.Elevated expression of CCT8 facilitates viral proliferation.

Abstract DNA and RNA are compartmentalized into distinct, heterogeneous structures within cells. However, the separation between RNA and DNA, especially in the absence of modern transcription machinery, is inherently hindered by thermodynamic constraints such as complementary base pairing and entropic mixing. Here, we demonstrate that atomic-level molecular difference between single-stranded DNA and RNA bearing identical sequences can drive the separation of DNA and RNA when complexed with peptides. This molecular mechanism is exploited to develop a family of oligonucleotides that enables DNA-RNA segregation within coacervates, resulting in a library of genetically encoded multiphase droplets containing coexisting DNA- and RNA-rich phases. These droplets emulate the structures and functions of multiphase nuclear compartments found in cells. Our results underscore the significance of pentose sugar mutation in DNA and RNA, which affect the genetic material organization in contemporary cells and could provide an evolutionary advantage during the transition from RNA to DNA genomes.

Influenza virus infection poses a great threat to human health globally each year. Non-coding RNAs (ncRNAs) in the human genome have been reported to participate in the replication process of the influenza virus, among which there are still many unknowns about Long Intergenic Non-Coding RNAs (LincRNAs) in the cell cycle of viral infections. Here, we observed an increased expression of Linc01615 in A549 cells upon influenza virus PR8 infection, accompanied by the successful activation of the intracellular immune system. The knockdown of Linc01615 using the shRNAs promoted the proliferation of the influenza A virus, and the intracellular immune system was inhibited, in which the expressions of IFN-β, IL-28A, IL-29, ISG-15, MX1, and MX2 were decreased. Predictions from the catRAPID website suggested a potential interaction between Linc01615 and DHX9. Also, knocking down Linc01615 promoted influenza virus proliferation. The subsequent transcriptome sequencing results indicated a decrease in Linc01615 expression after influenza virus infection when DHX9 was knocked down. Further analysis through cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) in HEK293 cells stably expressing DHX9 confirmed the interaction between DHX9 and Linc01615. We speculate that DHX9 may interact with Linc01615 to partake in influenza virus replication and that Linc01615 helps to activate the intracellular immune system. These findings suggest a deeper connection between DHX9 and Linc01615, which highlights the significant role of Linc01615 in the influenza virus replication process. This research provides valuable insights into understanding influenza virus replication and offers new targets for preventing influenza virus infections.

Japanese encephalitis is one of the most important insect-borne infectious disease with public health concern. The virus can break the blood–brain barrier and cause death or long-term sequela in infected humans or animals. Viral encephalitis is an important clinical feature of JEV infection. In recent studies, CircRNAs and related ceRNAs data illustrated the regulative role in many aspects of biological process and disease duration. It is believed that CircRNA regulates JEV infection in a ceRNA-dependent mechanism. In this study, brain tissues of experimental mice were sequenced and analysised. 61 differentially expressed circRNAs, 172 differentially expressed miRNAs and 706 differentially expressed mRNAs were identified by RNA-Sequencing and statistical analysis. CX3CR1 was determined as a key host factor impact JEV infection by microRNA interference measurement. CX3CR1 interaction network indicated circStrbp/miR709/CX3CR1 as a functional regulation axis. Further sequencing in BV2 cell shown CX3CR1 is a special target of miR-709 only during JEV infection. In summary, our study presented a new ceRNA pathway that impact JEV infection in vivo and in vitro, which could be a therapeutic target to fight against JEV.

Avian influenza (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2 alpha, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was 475LRGVRVSK482 and 528TITYSSPMMW537. Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits.

Mucosal immunity is the main defense line against respiratory disease pathogens. Newcastle disease and avian infectious bronchitis are common respiratory diseases in poultry. However, the mucosal immune response is not sufficiently activated and thus fails to achieve the ideal immune protection. Therefore, it is important to develop a suitable mucosal immune adjuvant to enhance the immune response of live vaccines. Here, the bursal-derived peptide BP7, β-glucan, and hyaluronic acid were selected as the adjuvant to be assembled into the composite nanopolypeptide adjuvant (CNPB7) with ultrasonic dispersion technology. The results showed that after optimizing assembly conditions, the optimal average particle size of nanoparticle CNPB7 was 514.9 nm and PDI was 0.298. To evaluate the non-specific immune responses of nanoparticle CNPB7, the chickens were immunized only with nanoparticle CNPB7. It was confirmed that nanoparticle CNPB7 enhanced the expression of CD3, CD4, CD80, and CD86 factors in the spleen lymphocyte from the chicken immunized with nanoparticle CNPB7. To investigate the mucosal immune response of nanoparticle CNPB7, the chickens were orally immunized with Newcastle disease virus (NDV)–infectious bronchitis virus (IBV) dual vaccines and CNPB7. The results proved that the levels of immunoglobulin SIgA, IL-4, IFN-γ, and IL-13 in the mucus samples from the respiratory and digestive tract in chicken immunized with nanoparticle CNPB7 and vaccines were significantly increased, compared to that of vaccine control. Finally, it was observed that nanoparticle CNPB7 promoted specific increased antibody productions against NDV and IBV in the immunized chicken. These results proved that the assembled nanoparticle CNPB7 could enhance the vaccination efficacy in chicken, which provided the experimental basis for the development of new adjuvants, and offered technical support for preventing virus transmission of avian diseases.

The Bursa of Fabricius, an avian unique humoral immune organ, is instrumental to B cell development. Bursal-derived peptide BP9 fosters B-cell development and formation. Yet, the exact mechanism wherein BP9 impacts B cell differentiation and antigenic presentation remains undefined. In this paper, B cell activation and differentiation in the spleen cells from mice immunized with the AIV vaccine and BP9 were detected following flow cytometry (FCM) analysis. Furthermore, the molecular mechanism of BP9 in B cell differentiation in vivo was investigated with RNA sequencing technology. To verify the potential functional mechanism of BP9 in the antigenic presentation process, the transcriptome molecular basis of chicken macrophages stimulated by BP9 was measured via high-throughput sequencing technology. The results proved that when given in experimental dosages, BP9 notably accelerated total B cells, and enhanced B-cell differentiation and plasma cell production. The gene expression profiles of B cells from mice immunized with 0.01 mg/mL BP9 and AIV vaccine disclosed that 0.01 mg/mL BP9 initiated the enrichment of several biological functions and significantly stimulated key B-cell pathways in immunized mice. Crucially, a total of 4093 differentially expressed genes were identified in B cells with BP9 stimulation, including 943 upregulated genes and 3150 downregulated genes. Additionally, BP9 induced various cytokine productions in the chicken macrophage HD11 cells and activated 9 upregulated and 20 downregulated differential miRNAs, which were involved in various signal and biological processes. Furthermore, BP9 stimulated the activation of multiple transcription factors in HD11 cells, which was related to antigen presentation processes. In summary, these results suggested that BP9 might promote B cell differentiation and induce antigen presentation, which might provide the valuable insights into the mechanism of B cell differentiation upon bursal-derived immunomodulating peptide stimulation and provide a solid experimental groundwork for enhancing vaccine-induced immunity.

In the original publication [...].

Recently, low pathogenic avian influenza virus (LPAIV), including H9N2 subtype, has been common clinical epidemic strains, and is widely distributed globally. The PB1 protein is a key component of the viral RNA polymerase complex (vRNP), and is vital to viral transcription and translation. In this study, to investigate the antigenic determinants in the PB1 protein, the truncated PB1 sequence (1bp-735bp) from H9N2 subtype AIV was amplified with PCR, and expressed in plasmid pET-28a (+). After purification, the recombinant PB1 protein was used to immunize BALB/c mice. Following immunization, hybridoma cells producing PB1-specific monoclonal antibodies were generated through the fusion of splenic lymphocytes with SP2/0 cells. Then, four stable hybridoma cell lines (5F12, 5B3, 2H9, and 3E6) were screened using indirect ELISA and Western blotting. Furthermore, two antigenic sites, 67NPIDGPLPED76 and 97ESHPGIFENS106, were identified through the construction of truncated overlapping fragments of the PB1 protein. These sites were conserved among 28 AIV strains, and were located on the PB1 protein surface. The findings offer a theoretical reference for the development and improvement of H9N2 vaccines and offer biological materials for virus detection during AIV infection mechanisms.

随着我国现代畜牧业健康养殖发展,对兽医专业人才的综合素质需求不断提升.动物药学(简称动药)专业本科生未来将承担兽药研发、生产、管理、服务等相关工作,兽用诊断与检测方面的专业课程教学对于增强其在动物疾病诊疗、检验检疫及初步科学研究能力等方面均具有深远影响.根据近 3 年来南京农业大学动物医学院动药专业《兽用诊断与检测制剂》课程教学实施情况,本文比较分析了该课程的教学效果,并从教学方法改进、理论知识、科研锻炼及突发传染病等方面阐述了动药专业本科生专业课程教学活动的影响因素及相应改进措施.总之,合理的专业课程教学模式不仅是优化课程教学活动、增强教学效果的重要手段,而且是提升动药专业本科生科研能力的重要途径,对我国动物生物制品行业的人才培养具有重要意义.

The infection and replication of avian influenza virus (AIV) in host cells is a complex biological process that involves the transport of viral genes through the host cell's transport systems. Actin, microtubules and vimentin are known to facilitate transport of endosomes to the perinuclear region, but the biological role of Keratin, another intermediate filament, in viral transport during AIV replication is not well understood. In this study, the viral NS2 protein was used as the target protein to identify the potential interacting proteins following GSTPulldown method and protein mass spectrometry. It was discovered that Keratin10 interacted with NS2. Subsequently, it was found AIV infection did not affect the gene level or protein level of keratin10 in HeLa cells, but when Keratin10 was knocked down, the expressions of viral NP mRNA and protein were reduced, and the generation of offspring virus also was also decreased. Furthermore, in early viral infection, Keratin10 could aggregate and co-localize with NP proteins, suggesting that Keratin10 might be connected to early viral transport. Additionally, it was demonstrated that Keratin10 co-localized with Lamp1 and that AIV particles were trapped in late endosomes/Lysosomes after Keratin10 was knocked down. Finally, it was discovered that the knocking down Keratin10 in HeLa cells led to an increase in the acidic pH of endosomes and lysosomes, which prevented AIV from undergoing fusion and uncoating, and then inhibited the process of the viral infection. Overall, the results suggested that Keratin10 might play the critical role in the release of vRNPs from LEs/Ls and can affect the generation of offspring virus. The study provides the novel insights into the role of Keratin10 in the process of AIV infection and transmission, which may have implications for developing new strategies to against AIV infections.

The oligos used for generation of sgRNA expression vectors

为了制备乙型脑炎病毒(Japanese encephalitis virus,JEV)结构蛋白PrM单克隆抗体,首先根据NCBI参考序列(GQ918133.2)设计PrM引物扩增目的基因,将目的基因构建至pCold Ⅰ原核表达质粒上;然后鉴定阳性克隆质粒并转化至BL21(DE3)感受态细胞,经诱导表达蛋白纯化后.免疫6周龄BALB/c小鼠,进行杂交瘤细胞融合,并对融合细胞进行克隆筛选;最后将筛选阳性的杂交瘤细胞制备腹水获取单克隆抗体,采用间接ELISA检测抗体效价,Western blot和间接免疫荧光检测抗体特异性和病毒感染过程.结果显示:成功构建了 pCold Ⅰ-PrM原核表达载体,PrM蛋白在1mmol/LIPTG 16 ℃过夜诱导培养时表达量较高,亚克隆后经腹水制备获得2株针对PrM区域的单克隆抗体,Western blot和间接免疫荧光检测2株单抗能够特异性识别JEV的PrM区蛋白,获得的单抗能够鉴定JEV在细胞内的感染过程.本研究为进一步研究JEV鉴别诊断技术和生物学功能提供基础.