Recombinant Structural Proteins Research Articles

Elasticity Modification of Biomaterials Used in 3D Printing with an Elastin-Silk-like Recombinant Protein.

The recombinant structural protein described in this study was designed based on sequences derived from elastin and silk. Silk-elastin hybrid copolymers are characterized by high solubility while maintaining high product flexibility. The phase transition temperature from aqueous solution to hydrogel, as well as other physicochemical and mechanical properties of such particles, can differ significantly depending on the number of sequence repeats. We present a preliminary characterization of the EJ17zipR protein obtained in high yield in a prokaryotic expression system and efficiently purified via a multistep process. Its addition significantly improves biomaterial's rheological and mechanical properties, especially elasticity. As a result, EJ17zipR appears to be a promising component for bioinks designed to print spatially complex structures that positively influence both shape retention and the internal transport of body fluids. The results of biological studies indicate that the addition of the studied protein creates a favorable microenvironment for cell adhesion, growth, and migration.

Natural Antibodies Produced in Vaccinated Patients and COVID-19 Convalescents Hydrolyze Recombinant RBD and Nucleocapsid (N) Proteins.

Antibodies are protein molecules whose primary function is to recognize antigens. However, recent studies have demonstrated their ability to hydrolyze specific substrates, such as proteins, oligopeptides, and nucleic acids. In 2023, two separate teams of researchers demonstrated the proteolytic activity of natural plasma antibodies from COVID-19 convalescents. These antibodies were found to hydrolyze the S-protein and corresponding oligopeptides. Our study shows that for antibodies with affinity to recombinant structural proteins of the SARS-CoV-2: S-protein, its fragment RBD and N-protein can only hydrolyze the corresponding protein substrates and are not cross-reactive. By using strict criteria, we have confirmed that this proteolytic activity is an intrinsic property of antibodies and is not caused by impurities co-eluting with them. This discovery suggests that natural proteolytic antibodies that hydrolyze proteins of the SARS-CoV-2 virus may have a positive impact on disease pathogenesis. It is also possible for these antibodies to work in combination with other antibodies that bind specific epitopes to enhance the process of virus neutralization.

Biological effects and mechanism of β-amyloid aggregation inhibition by penetrable recombinant human HspB5-ACD structural domain protein

Alzheimer's disease (AD) is a global medical challenge. Studies have shown that neurotoxicity caused by pathological aggregation of β-amyloid (Aβ) is an important factor leading to AD. Therefore, inhibiting the pathological aggregation of Aβ is the key to treating AD. The recombinant human HspB5-ACD structural domain protein (AHspB5) prepared by our group in the previous period has been shown to have anti-amyloid aggregation effects, but its inability to penetrate biological membranes has limited its development. In this study, we prepared a recombinant fusion protein (T-AHspB5) of TAT and AHspB5. In vitro experiments showed that T-AHspB5 inhibited the formation of Aβ1–42 protofibrils and had the ability to penetrate the blood-brain barrier; in cellular experiments, T-AHspB5 prevented Aβ1–42-induced oxidative stress damage, apoptosis, and inflammatory responses in neuronal cells, and its mechanism of action was related to microglia activation and mitochondria-dependent apoptotic pathway. In animal experiments, T-AHspB5 improved memory and cognitive dysfunction and inhibited pathological changes of AD in APP/PS1 mice. In conclusion, this paper is expected to reveal the intervention mechanism and biological effect of T-AHspB5 on pathological aggregation of Aβ1–42, provide a new pathway for the treatment of AD, and lay the foundation for the future development and application of T-AHspB5.

Organized assembly of chitosan into mechanically strong bio-composite by introducing a recombinant insect structural protein OfCPH-1

Chitosan, known for its appealing biological properties in packaging and biomedical applications, faces challenges in achieving a well-organized crystalline structure for mechanical excellence under mild conditions. Herein, we propose a facile and mild bioengineering approach to induce organized assembly of amorphous chitosan into mechanically strong bio-composite via incorporating a genetically engineered insect structural protein, the cuticular protein hypothetical-1 from the Ostrinia furnacalis (OfCPH-1). OfCPH-1 exhibits high binding affinity to chitosan via hydrogen-bonding interactions. Simply mixing a small proportion (0.5 w/w%) of bioengineered OfCPH-1 protein with acidic chitosan precursor induces the amorphous chitosan chains to form fibrous networks with hydrated chitosan crystals, accompanied with a solution-to-gel transition. We deduce that the water shell destruction driven by strong protein-chitosan interactions, triggers the formation of well-organized crystalline chitosan, which therefore offers the chitosan with significantly enhanced swelling resistance, and strength and modulus that outperforms that of most reported chitosan-based materials as well as petroleum-based plastics. Moreover, the composite exhibits a stretch-strengthening behavior similar to the training living muscles on cyclic load. Our work provides a route for harnessing the OfCPH-1-chitosan interaction in order to form a high-performance, sustainably sourced bio-composite.

Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia

BackgroundAlthough E. coli is generally a well-opted platform for the overproduction of recombinant antigens as heterologous proteins, the optimization of expression conditions to maximize the yield of functional proteins remains empirical. Herein, we developed an optimized E. coli (BL21)-based system for the overproduction of soluble immunoreactive HCV core/envelope proteins that were utilized to establish a novel immunoassay for discrimination of active HCV infection.MethodsThe core/E1-E2 genes were amplified and expressed in E. coli BL21 (DE3) in the absence/presence of glycylglycine. The antigenic performance of soluble proteins was assessed against 63 HCV-seronegative (Ab−) sera that included normal and interferent sera (HBV and/or chronic renal failure), and 383 HCV-seropositive (Ab+) samples that included viremic (chronic/relapsers) and recovered patients’ sera. The color intensity (OD450) and S/Co values were estimated.ResultsThe integration of 0.1–0.4M glycylglycine in the growth media significantly enhanced the solubility/yield of recombinant core and envelope proteins by ~ 225 and 242 fold, respectively. This was reflected in their immunoreactivity and antigenic performance in the developed immunoassay, where the soluble core/E1/E2 antigen mixture showed 100% accuracy in identifying HCV viremic sera with a viral RNA load as low as 3800 IU/mL, without cross-reactivity against normal/interferent HCV-Ab−sera. The ideal S/Co threshold predicting active viremia (> 2.75) showed an AUC value of 0.9362 (95% CI: 0.9132 to 0.9593), with 87.64, 91.23% sensitivity and specificity, and 94.14, 82.11% positive and negative predictive values, respectively. The different panels of samples assayed with our EIA showed a good concordance with the viral loads and also significant correlations with the golden standards of HCV diagnosis in viremic patients. The performance of the EIA was not affected by the immunocompromised conditions or HBV co-infection.ConclusionThe applicability of the proposed platform would extend beyond the reported approach, where glycylglycine, low inducer concentration and post-induction temperature, combined with the moderately-strong constitutive promoter enables the stable production of soluble/active proteins, even those with reported toxicity. Also, the newly developed immunoassay provides a cost-effective point-of-care diagnostic tool for active HCV viremia that could be useful in resource-limited settings.

Recombinant human HspB5-ACD structural domain inhibits neurotoxicity by regulating pathological α-Syn aggregation

The treatment of Parkinson's disease is a global medical challenge. α-Synuclein (α-Syn) is the causative protein in Parkinson's disease and is closely linked to its progression. Therefore, inhibiting the pathological aggregation of α-Syn and its neurotoxicity is essential for the treatment of Parkinson's disease. In this study, α-Syn and recombinant human HspB5-ACD structural domain protein (AHspB5) were produced using the BL21(DE3) E. coli prokaryotic expression system, and then the role and mechanism of AHspB5 in inhibiting the pathological aggregation of α-Syn and its neurotoxicity were investigated. As a result, we expressed α-Syn and AHspB5 proteins and characterised the proteins. In vitro experiments showed that AHspB5 could inhibit the formation of α-Syn oligomers and fibrils; in cellular experiments, AHspB5 could prevent α-Syn-induced neuronal cell dysfunction, oxidative stress damage and apoptosis, and its mechanism of action was related to the TH-DA pathway and mitochondria-dependent apoptotic pathway; in animal experiments, AHspB5 could inhibit behavioural abnormalities, oxidative stress damage and loss of dopaminergic neurons. In conclusion, this work is expected to elucidate the mechanism and biological effects of AHspB5 on the pathological aggregation of α-Syn, providing a new pathway for the treatment of Parkinson's disease and laying the foundation for recombinant AHspB5.

Biosynthesis, Assembly, and Biomedical Applications of High-Performance Engineered Proteins.

Engineered structural proteins, mimicking the structure and function of well-characterized natural proteins, are of great interest for diverse applications due to their outstanding mechanical performance and hierarchical structures. Broad efforts have been devoted to developing novel toolsets of genetically engineered structural proteins to explore advanced protein-based materials. With the rational design and structural optimization of artificially structural proteins and the improved biosynthetic methods, artificial protein assemblies have demonstrated outstanding mechanical performance comparable to those of natural protein materials, showing promising biomedical applications. In this Review, we outline recent advances in the fabrication of high-performance protein materials, highlighting the roles of biosynthesis, structural modification, and assembly in optimizing the materials' properties. The relationship between hierarchical structures and the mechanical performance of these recombinant structural proteins is discussed in detail. We emphasize the biomedical applications of high-performance structural proteins and their assemblies in the fields of high-strength protein fibers and adhesives. Finally, we discuss the trends and perspectives for the development of structural protein-based materials.

Functional characterization and immunogenicity of a novel vaccine candidate against tick-borne encephalitis virus based on Leishmania-derived virus-like particles

Tick-borne encephalitis virus (TBEV) is a major cause of neurological infections in many regions of central, eastern and northern Europe and northern Asia. In approximately 15% of cases, TBEV infections lead to the development of severe encephalitis or meningitis. The main route of TBEV transmission is tick bites; however, ingestion of dairy products from infected animals (goats, cattle and sheep) is also a frequent cause of the disease. Therefore, vaccination of livestock in virus endemic regions could also contribute to the decrease in TBEV infection among humans. Although few vaccines against TBEV based on inactivated viruses are available for humans, due to high costs, vaccination is not mandatory in most of the affected countries. Moreover, there is still no vaccine for veterinary use. Here, we present a characterization and immunogenicity study of a new potential TBEV vaccine based on virus-like particles (VLPs) produced in Leishmania tarentolae cells. VLPs, which mimic native viral particles but do not contain genetic material, show good immunogenic potential. For the first time, we showed that the protozoan L. tarentolae expression system can be successfully used for the production of TBEV virus-like particles with highly efficient production. We confirmed that TBEV recombinant structural proteins (prM/M and E) from VLPs are highly recognized by neutralizing antibodies in in vitro analyses. Therefore, VLPs in combination with AddaVax adjuvant were used in immunization studies in a mouse model. VLPs proved to be highly immunogenic and induced the production of high levels of neutralizing antibodies. In a challenge experiment, immunization with VLPs provided full protection from lethal TBE in mice. Thus, we suggest that Leishmania-derived VLPs may be a good candidate for a safe alternative human vaccine with high efficiency of production. Moreover, this potential vaccine candidate may constitute a low-cost candidate for veterinary use.

Molecular characterization of recombinant premembrane protein of dengue virus serotype-2 for development of diagnostic assay.

Dengue is an acute arboviral infection common in tropical and subtropical countries. Dengue has been highlighted as a public health concern in the last five decades, affecting almost 50% of the population in developing nations. Dengue infection results in a complex symptomatic disease that ranges from headache, fever, and skin rash to extreme hemorrhage fever and liver dysfunction. The diagnosis of the disease is essential for effective treatment. The early onset of the infection can be detected through viral structural peptides that act as markers for detection, including Pre-Membrane (Pre-M) protein. In the currently proposed research, the structural gene obtained from local isolates was targeted for studies. For this purpose, recombinant structural protein Pre-M was amplified, cloned, and expressed in the bacterial expression system. The expression of the structural protein (Pre-M) was scrutinized by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and validated by western blot and dot blot, and afterwards, the antigen was purified. The purified Pre-M protein carries the potential for the development of in-house diagnostic assay as well as for vaccine production. This study aimed to develop a highly specific, sensitive, and cost-effective in-house enzyme-linked immunoassay (ELISA) for the detection of antibodies of Pakistani most prevalent dengue virus serotype 2 (DENV-2). The success of this research would also pave the way toward developing novel vaccines for the future prevention of dengue infection.

Scalable purification of recombinant structural proteins, hagfish intermediate filament α and ɣ, from inclusion bodies for fiber formation

The purpose of this study was to determine a method to purify recombinant hagfish intermediate filament proteins, alpha and gamma, in a scalable manner. The study succeeded by having an increase in protein recovery of up to 35% when comparing centrifuge purification and the developed tangential flow purification. The proteins were approximately the same purity of 70% pure but further purification increased the purity of the proteins by 16%, based on ImageJ analysis. The developed tangential flow filtration purification and final purification methods could be easily scaled up to meet industry scale purification needs. The scaled-up processes described in this study did not interfere with fiber production or formation, indicating the methods can produce usable proteins for material development.

Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a devastating disease of domestic and wild pigs. There is no effective vaccine, and the control of the disease relies mainly on surveillance and early detection of infected pigs. Previously, serological assays, such as ELISA, have been developed mainly based on recombinant structural viral proteins of ASFV, including p72, p54, and p30. However, the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs. Therefore, new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required. In this study, we expressed and purified a recombinant pB602L protein. The purified pB602L protein was then used as an antigen to develop an indirect ELISA assay. This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China, such as classical swine fever virus, pseudorabies virus, and porcine parvovirus. This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples, including an unknown number of anti-ASFV sera. The coincidence of the two assays was 95%. Furthermore, the pB602L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7GD in pigs. The results showed that the antibody levels in all vaccinated pigs, starting from the 10th day post-inoculation, have increased continuously during the observation period of 45 days. Our results indicate that this pB602L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

WITHDRAWN: Development and evaluation of an indirect ELISA based on recombinant structural protein σC to detect antibodies against avian orthoreovirus

The avian orthoreovirus (ARV) causes large economic losses to poultry industry around the whole world. ARV viral proteins can be classified into three sizes: λ (large), μ (medium) and σ (small). σC, one of the capsid protein of ARV, contains various specific neutralizing epitopes and can induce robust immune responses in infected chickens. An indirect enzyme-linked immunosorbent assay (I-ELISA) was established using the recombinant σC protein. The optimal dilutions of antigen, serum and conjugate goat anti-chicken IgY conjugate were 2.5 μg/ml, 1:80 and 1:4000, respectively. The optimal antigen coating condition was 4℃ for overnight. Hyperimmune serum of high-yielding hens of different age produced stronger reaction against σC. Chicken serum samples were randomly collected and total coincidence rate between commercial kit and σC-ARV-ELISA kit was 97.8%. Importantly, those discrepant serums were further verified using Western blot assay and three of them were determined to be positive. Thus, the method based on σC protein has higher sensitivity compared with the commercial kit and provide a rapid and simple method for serodiagnosis of ARV, contributing to convenient epidemiological monitoring of ARV.

Detections of Rainbow Trout Antibodies to Infectious Pancreatic Necrosis Virus and Viral Hemorrhagic Septicemia Virus via a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay

Infectious Pancreatic Necrosis Virus (IPNV) and Viral hemorrhagic septicemia virus (VHSV) cause significant losses in the aquaculture industry. There have been few reports of the use of screening rainbow trout for antibodies against IPNV and VHSV as an epidemiological tool. Several ELISAs using a whole virus or recombinant IPNV and VHSV proteins have been described. In this study, a recombinant protein-based enzyme-linked immunosorbent assay (ELISA) for the detection of IPNV and VHSV antibodies in rainbow trout (Oncorhynchus mykiss) was evaluated. To develop recombinant protein-based enzyme-linked immunosorbent assays, a fragment containing the entire length of the gG gene of VHSV and VP2 of IPNV was amplified by PCR using the viruses' genomic RNA and cloned in pET-28a(+) plasmid. Recombinant structural viral proteins (rVP2 and rgG) were expressed in the Escherichia coli BL21 (DE3). The rgG was extracted and purified. 96-well plates were coated with VP2 and gG separately. For VHSV, Assay could detect until 1/15625 dilution in VHSV positive fish serum. For IPNV, Assays could detect until 1/3125 dilution in IPNV positive fish serum. These results show rgG and rVP2, used in ELISA, are more sensitive than virus neutralization tests.

SARS-CoV2 spike protein induces inflammatory molecules through TLR2 in macrophages and monocytes

Abstract COVID-19 pandemic caused by SARS-CoV2 infection has emerged as a major public health problem. Excessive inflammation or cytokine storm is considered a major cause of respiratory failure, organ damage, and death of COVID-19 patients. However, the precise mechanism of inflammatory response induced by SARS-CoV2 is poorly understood. We investigated whether the structural proteins of SARS-CoV2 including spike (S), membrane (M), envelope (E), and neucleocapsid (N) are involved in inducing inflammation. Human monocytic cells THP-1 derived macrophages or human peripheral blood mononuclear cells were stimulated with the recombinant structural proteins and measured for the induction of inflammatory cytokines and chemokines. We observed that S protein potently induces IL-6, IL-1b, TNFa, CXCL1, and CCL2 in macrophages and monocytes, while other structural proteins were non-stimulatory. Notably, human lung epithelial cells A549 and human embryonic kidney epithelial cell HEK293T, which express S protein receptor ACE2, did not express cytokines and chemokines in response to S and other structural proteins. We dissected signaling pathways involved in inducing inflammatory molecules in response to S protein. S protein activates the NF-kB pathway in a MyD88 dependent manner. Further, S protein-mediated activation of NF-kB and induction of inflammatory responses were completely abrogated in TLR2-deficient macrophages, suggesting that TLR2 is an innate immune receptor for SARS-CoV2 S protein. Together these data reveal a mechanism of cytokine storm during SARS-CoV2 infection, and points to TLR2 as a potential therapeutic target for COVID-19.

Environmental biodegradability of recombinant structural protein

Next generation polymers needs to be produced from renewable sources and to be converted into inorganic compounds in the natural environment at the end of life. Recombinant structural protein is a promising alternative to conventional engineering plastics due to its good thermal and mechanical properties, its production from biomass, and its potential for biodegradability. Herein, we measured the thermal and mechanical properties of the recombinant structural protein BP1 and evaluated its biodegradability. Because the thermal degradation occurs above 250 °C and the glass transition temperature is 185 °C, BP1 can be molded into sheets by a manual hot press at 150 °C and 83 MPa. The flexural strength and modulus of BP1 were 115 ± 6 MPa and 7.38 ± 0.03 GPa. These properties are superior to those of commercially available biodegradable polymers. The biodegradability of BP1 was carefully evaluated. BP1 was shown to be efficiently hydrolyzed by some isolated bacterial strains in a dispersed state. Furthermore, it was readily hydrolyzed from the solid state by three isolated proteases. The mineralization was evaluated by the biochemical oxygen demand (BOD)-biodegradation testing with soil inocula. The BOD biodegradability of BP1 was 70.2 ± 6.0 after 33 days.

Development and evaluation of an indirect ELISA based on recombinant structural protein VP2 to detect antibodies against duck hepatitis A virus

An indirect enzyme-linked immunosorbent assay (I-ELISA) based on the VP2 protein of duck hepatitis A virus type 3 (DHAV-3) was established in this study. The optimal dilutions of antigen, serum and goat anti-duck IgG conjugate were 1:1600 (2.23 μg/mL), 1:160 and 1:2000, respectively. The optimal blocking buffer was 1% skim milk. The cut-off value for the method was 0.25, and the analytical sensitivity of the method was 1:5120. The results of specific evaluation showed that except for DHAV-1, DHAV-3 antisera did not cross-react with any other common duck-sensitive pathogens, indicating that this method can be used to detect DHAV-3 and DHAV-1 antibodies. The coefficients of variation (CVs) were lower than 10 %. The coincidence rate between the VP2-DHAV-3-ELISA and the neutralization test was 93.3 %. In summary, the I-ELISA method based on VP2 protein has high sensitivity, specificity, and coincidence rate compared with the neutralization test and has advantages in serum monitoring. The I-ELISA method based on VP2 protein provides a simple and rapid method for the detection of anti-DHAV antibodies and the epidemiological monitoring of DHAV.

Directed Evolution of Structural Proteins using a High Throughput Approach

Design of novel de novo structural proteins from their primary amino acid sequence is an unsolved scientific problem. Moreover, most techniques that are used for predicting the protein structure rely on globular protein-structure databases and are rarely applicable to structural-protein assemblies. Experimental characterization for this class of proteins often employs spectroscopic techniques including but not limited to Fourier transform infrared spectroscopy (FTIR), X-ray crystallography, Raman spectroscopy, or light scattering. Furthermore, existing high-throughput fluorescence and affinity readouts are structure-specific and may miss materials with novel molecular morphologies. We developed a network model based on entropic elasticity to predict structure-property relationship for proteins, and studied the structure-property relationship of these engineered proteins using ultrafast laser-probing spectroscopy. We performed time domain transient transmissivity experiments using purified recombinant structural proteins, which clearly showed “proof of concept” quantification of protein properties in real time. This technique will help us to identify structural proteins that have the ability to assemble and form cross-linked biomaterials.

Biocompatibility and immunogenic response to recombinant honeybee silk material.

If tolerated in biological environments, recombinant structural proteins offer the advantage that biological cues dictating cell attachment and material degradation can be modified as required for clinical application using genetic engineering. In this study, we investigate the biological response to materials generated from the recombinant honeybee silk protein, AmelF3, a structural protein that can be produced at high levels by fermentation in Escherichia coli. The protein can be readily purified from E. coli host cell proteins after transgenic production and fabricated into various material formats. When implanted subcutaneously according to International Standard ISO 10993 tests, materials generated from the purified recombinant protein were found to be noncytotoxic, inducing a transient weak immunogenic response and a chronic inflammatory response that resolved over time. While preliminary, this study supports the ongoing development of materials generated from this protein for biomedical applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1763-1770, 2019.

An efficient production of hybrid recombinant protein comprising non-structural proteins (NS 1 & NS 3) of bluetongue virus in prokaryotic expression system

Strategic design and suitable purification techniques are of paramount importance in the production of recombinant proteins, if intended for use in a diagnostic assay. However, there is no single protocol that can be universally adopted for obtaining proteins in requisite quality and quantity across various platforms. In this study, we have targeted proteins of bluetongue virus (BTV), which is the causative agent of an arthropod-borne infectious disease in ruminants. Traditionally, serological diagnosis of the disease has rested upon either virus neutralization test or on an ELISA test that employed a recombinant structural (VP1, VP7) protein. Among the non-structural (NS) proteins of BTV, NS1 and NS3, are preferred candidate antigens in development of immuno-diagnostics as these provide the option for identifying recent/ongoing infection. However, the difficulty in production/purification of recombinant full length NS proteins of BTV in sufficient quantity and quality in various expression systems, due to inherent structural complexities, have restricted their wider applicability as immunodiagnostic reagents. To circumvent the difficulties associated with production/purification, we developed a novel NS1 and NS3 fusion gene (∼1302 bp) encoding for NS1 N-terminus (1M to G252 aa) and NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains along with intervening variable central domain (118A to A182 aa) of bluetongue virus 23. This construct was cloned, over-expressed and efficiently purified by single step affinity chromatography under unique denaturing/renaturing condition. The purified fusion protein was found suitable for detection of antibodies against BTV in an indirect ELISA (iELISA).

Duck hepatitis A virus structural proteins expressed in insect cells self-assemble into virus-like particles with strong immunogenicity in ducklings

Duck hepatitis A virus (DHAV), a non-enveloped ssRNA virus, can cause a highly contagious disease in young ducklings. The three capsid proteins of VP0, VP1 and VP3 are translated within a single large open reading frame (ORF) and hydrolyzed by protease 3CD. However, little is known on whether the recombinant viral structural proteins (VPs) expressed in insect cells could spontaneously assemble into virus-like particles (VLPs) and whether these VLPs could induce protective immunity in young ducklings. To address these issues, the structural polyprotein precursor gene P1 and the protease gene 3CD were amplified by PCR, and the recombinant proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures and immunogenicity. The recombinant proteins expressed in Sf9 cells were detected by indirect immunofluorescence assay and Western blot analysis. Electron microscopy showed that the recombinant proteins spontaneously assembled into VLPs in insect cells. Western blot analysis of the purified VLPs revealed that the VLPs were composed with the three structural proteins. In addition, vaccination with the VLPs induced high humoral immune response and provided strong protection. Therefore, our findings may provide a framework for development of new vaccines for the prevention of duck viral hepatitis.