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OA

The Japanese eel(Anguilla japonica)is a globally important aquaculture species,but intensive farming has led to frequent viral diseases threatening its sustainability.Eel circovirus(EeCV),a key pathogen,causes immunosuppression,secondary infections,and major economic losses.EeCV is a small single-stranded circular DNA virus with a 1,378 bp genome encoding replication-associated(Rep)and capsid(Cap)proteins.This study reports the first detection of a circovirus in seawater-farmed Japanese eels,designating the viral genome as AJCVXM(PV393740).Current EeCV research focuses on diagnostic methods,leaving gaps in understanding Cap protein functions and developing efficient diagnostics. 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 using Western Blot,providing essential biological materials for future functional studies on the AJCVXM Cap protein and the development of immunological detection methods(e.g.,indirect ELISA). AJCVXM was detected in seawater-farmed Japanese eels,and its full Cap coding region(345 bp)was amplified.Wild-type Cap(345 bp),codon-optimized Cap(eCap,345 bp),and NLS-deleted Cap(eCap(N),243 bp)were analyzed.Sequence alignment with European EeCV Cap protein(APZ87903.1)revealed 92.98％homology,with 14 nucleotide differences resulting in 8 amino acid substitutions.The three Cap variants were cloned into the pET-28a vector to enhance prokaryotic expression,generating recombinant plasmids pET28a-Cap/eCap/eCap(N).Protein expression was compared under standard conditions(37 ℃,0.5 mmol/L IPTG)to identify the highest-yielding construct.Optimization experiments tested different media(LB,TB),induction temperatures(16 ℃,25 ℃,37 ℃),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 using indirect ELISA:the purified eCap(N)(10 μg/mL)was coated overnight,serially diluted antibodies(1∶1,000-1∶128,000)were incubated(37 ℃,2 h),and HRP-conjugated secondary antibodies were used for detection(TMB substrate,OD450 nm).Antibody specificity was validated using Western Blotting:recombinant eCap(N)(13.8 kDa)and a negative control(pET28a-nphp4,33.2 kDa)were separated using SDS-PAGE,transferred to PVDF membranes,and probed with the polyclonal antibody(1∶2,000).Tissue proteins from AJCVXM-infected and healthy eel gills were extracted,quantified(BCA assay),and analyzed. The current challenges in EeCV research involve the inability to propagate the virus through cell culture or injection,which complicates vaccine development.Prokaryotic expression systems,such as E.coli,provide a feasible alternative for the production of recombinant Cap proteins.However,the presence of arginine residues within the NLS-rich region and rare codons within the Cap gene hinders 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 pET28a-Cap plasmid showed no detectable expression even after codon optimization.Structural analysis predicted the NLS region within AJCVXM Cap(6-34 aa).Comparative expression of pET28a-Cap/eCap/eCap(N)revealed that NLS removal significantly improved protein yield in E.coli.Under optimal conditions(TB medium,0.1 mmol/L IPTG,37 ℃,5 h),the eCap(N)yield reached 9.78 mg/L,primarily as inclusion bodies.SDS-PAGE and Western Blot analyses confirmed the size(13.8 kDa)and specificity of the purified protein.Notably,the AJCVXM Cap differs from other EeCV Caps by eight amino acids,which may explain the divergent expression efficiencies.Transmission electron microscopy confirmed that eCap(N)could self-assemble into VLPs,although the assembly 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(OD450nm＞2.1)at a dilution of 1∶32,000.Specificity tests verified that the antibody specifically bound recombinant eCap(N)and AJCVXM-infected tissues,indicating its diagnostic potential. 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 research establishes a technical foundation for future functional studies and outbreak management in eel aquaculture.