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| 日本鳗鲡圆环病毒衣壳蛋白的原核表达和多克隆抗体制备 |
| Identification and Detection Technology Research of Circovirus in Anguilla japonica |
| 投稿时间：2025-04-17 修订日期：2025-05-15 |
| DOI： |
| 中文关键词: 日本鳗鲡 圆环病毒 衣壳蛋白 多克隆抗体 |
| 英文关键词: Anguilla japonica circovirus Cap protein Polyclonal antibody |
| 基金项目:国家重点研发计划(2024YFD2401003)；集美大学科研(ZQ2020003)作者简历班宇(1999--),女,硕士生,研究方向为鱼类抗病分子育种。Email: 202212951057@jmu.edu.cn 通讯作者陈天圣(1977--),男,博士,教授,研究方向为鱼类遗传育种。Email: tiansheng.chen@jmu.edu.cn |
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| 中文摘要: |
| 【目的】鳗鲡圆环病毒(Eel circovirus,EeCV)的衣壳蛋白(Cap)是病毒感染诊断与疫苗研发的重要抗原。本研究旨在利用原核表达系统实现重组Cap蛋白在大肠杆菌中高效表达并制备多克隆抗体。【方法】从海水养殖日本鳗鲡(Anguilla japonica)样本中检测到鳗鲡圆环病毒,并扩增了完整的Cap编码区(345 bp),并分别将Cap基因、经大肠杆菌密码子偏好性优化后的Cap基因(eCap,345 bp)以及去核定位信号(Nuclear localization sequence,NLS)的Cap基因(eCap(N),243 bp)均克隆至pET-28a表达载体中构建出高表达的重组质粒。优化诱导培养基和IPTG终浓度后进行大量表达纯化以及动物免疫实验制备多克隆抗体,并对该抗体进行效价检测和特异性鉴定。【结果】成功构建出高表达的重组质粒pET28a-eCap(N),其目的蛋白分子量为13.8 kDa；在37℃,TB肉汤培养基(Terrific Broth),IPTG终浓度0.1 mmol/L条件下诱导5 h后目的蛋白以包涵体形式存在；经纯化透析复性在1L培养基中获得9.78 mg的eCap(N)蛋白；制备的多克隆抗体与抗原结合最大稀释倍数为1：32000,显示出良好的免疫原性；且Western Blot结果显示该多克隆抗体可以特异性识别重组表达的目的蛋白与感染鳗鲡圆环病毒的样本。【结论】本研究成功制备抗Cap多克隆抗体,为后续深入研究Cap蛋白功能及EeCV血清学诊断方法与疫苗等相关生物制品研发奠定基础。 |
| 英文摘要: |
| Abstract: 【Background】The Japanese eel (Anguilla japonica) is a globally significant aquaculture species. However, the increasing adoption of intensive farming practices has led to frequent viral outbreaks that severely threaten the sustainability of the industry. Among these pathogens, eel circovirus (EeCV), previously identified in European eels (Anguilla anguilla) and American eels (Anguilla rostrata), often causes immunosuppression in infected hosts, leading to concurrent secondary infections and substantial economic losses. EeCV is a small single-stranded circular DNA virus with a genome of 1,378 bp. Its compact genome contains two major open reading frames (ORFs) encoding the replication-associated protein (Rep, 286 aa) and the capsid protein (Cap, 114 aa).This study reports the first detection of a circovirus in seawater-farmed Japanese eels. The sequenced and assembled viral genome was designated AJCVXM (Anguilla japonica Circovirus Xiamen, PV393740). Current research on eel circoviruses primarily focuses on diagnostic method development, leaving significant gaps in understanding Cap protein functions and the creation of high-efficiency diagnostic tools. The Cap protein plays a central role in viral particle assembly and host cell invasion. However, its expression in prokaryotic systems remains challenging. Codon optimization is widely employed to enhance the expression efficiency of exogenous genes in prokaryotic hosts. For instance, codon optimization of the porcine circovirus type 3 (PCV3) Cap gene significantly improved its soluble expression in E. coli. Additionally, the nuclear localization signal (NLS), responsible for protein transport from the cytoplasm to the nucleus, may not be critical for conformational epitopes. When the NLS of the Cap protein contains abundant arginine residues, its hydrophobicity increases, likely hindering full-length Cap expression in prokaryotic systems.
【Objective】This study aims to optimize the cloning of the AJCVXM Cap gene, identify highly expressed recombinant Cap protein, and produce polyclonal antibodies through large-scale expression and purification. The specificity of these antibodies will be validated via Western blotting, providing essential biological materials for future functional studies of the AJCVXM Cap protein and the development of immunological detection methods (e.g., indirect ELISA).
【Methods】AJCVXM was detected in seawater-farmed Japanese eels, and its full Cap coding region (345 bp) was amplified. Three Cap gene variants were analyzed: the wild-type Cap, a codon-optimized Cap (eCap, 345 bp), and an NLS-deleted Cap (eCap(N), 243 bp). Sequence alignment with the European eel circovirus Cap protein (APZ87903.1) revealed 92.98% homology, with 14 nucleotide differences resulting in 8 amino acid substitutions.To enhance prokaryotic expression, the three Cap variants were cloned into the pET-28a vector, generating recombinant plasmids pET28a-Cap/eCap/eCap(N). Protein expression under standard conditions (37°C, 0.5 mmol/L IPTG) was compared to identify the highest-yielding construct. Optimization experiments tested different media (LB, TB), induction temperatures (16°C, 25°C, 37°C), and IPTG concentrations (0.1–1.5 mmol/L). Following optimization, large-scale expression (500 mL culture), purification, and rabbit immunization were performed.Antibody titers were assessed via indirect ELISA: purified eCap(N) (10 μg/mL) was coated overnight, serially diluted antibodies (1:1000–1:128,000) were incubated (37°C, 2 h), and HRP-conjugated secondary antibodies were used for detection (TMB substrate, OD450 measurement).Antibody specificity was validated by Western blotting: recombinant eCap(N) (13.8 kDa) and a negative control (pET28a-nphp4, 33.1 kDa) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with the polyclonal antibody (1:2000). Tissue proteins from AJCVXM-infected and healthy eel gills were extracted, quantified (BCA assay), and analyzed similarly.【Results】The current challenges in eel circovirus research involve the inability to propagate the virus through cell culture or injection, which complicates the development of vaccines using whole-virus antigens. Prokaryotic expression systems, such as E. coli, provide a feasible alternative for recombinant Cap protein production. However, the presence of nuclear localization signal (NLS)-rich arginine residues and rare codons within the Cap gene hinder high-yield prokaryotic expression. Studies on porcine and duck circoviruses have demonstrated that NLS deletion enhances Cap expression and enables self-assembly into virus-like particles (VLPs), which exhibit strong immunogenicity and diagnostic potential.In preliminary experiments, the plasmid pET28a-Cap showed no detectable expression even after codon optimization. Structural analysis predicted the NLS region within AJCVXM Cap (amino acids 6–34 aa). Comparative expression of pET28a-Cap/eCap/eCap(N) revealed that removal of the NLS significantly improved protein yield in E. coli. Under optimal conditions (TB medium, 0.1 mmol/L IPTG, 37°C, 5 h), the yield of eCap(N) reached 9.78 mg/L, primarily as inclusion bodies. SDS-PAGE and Western blot analyses confirmed the purified protein's size (13.8 kDa) and specificity. Notably, the AJCVXM Cap differs from other eel circovirus Caps by eight amino acids, which may explain the divergent expression efficiencies observed.Transmission electron microscopy confirmed that eCap(N) could self-assemble into VLPs, although their efficiency was lower than previously reported EeCV VLPs, potentially due to inclusion body purification or NLS truncation. Polyclonal antibodies were generated by immunizing rabbits with purified eCap(N). Antibody titer analysis revealed a positive signal (OD450 ratio >2.1) at a dilution of 1:32,000, confirming an antibody titer of 1:32,000. Specificity tests demonstrated that the antibody selectively recognized both recombinant eCap(N) and tissues infected with AJCVXM, supporting its potential for diagnostic applications.【Conclusion】This study successfully expressed an NLS-deleted AJCVXM Cap protein in E. coli and generated highly specific polyclonal antibodies with a titer of 1:32,000. These results provide essential materials for developing ELISA-based diagnostics, evaluating subunit vaccine immunogenicity, and investigating AJCVXM pathogenesis. This work establishes a technical foundation for future functional studies and outbreak management in eel aquaculture. |
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