Production and purification of immunogenic virus-like particles formed by the chimeric infectious bursal disease virus structural protein,rVP2H,in insect larvae

This study describes an alternative approach to produce rVP2H protein using insect larvae of the cabbage looper Trichoplusia ni as hosts for the expression of the protein. The chimeric rVP2H protein, having an extra six histidine residues at the C-terminus of the VP2, a structural protein of infectious bursal disease virus (IBDV), is a vaccine candidate for the prevention of infectious bursal disease. The chimeric rVP2H protein was expressed in insect larvae in form of virus-like particles, in which they maintain their native immunogenic properties. The expression level of rVP2H protein in T. ni larvae was estimated to be approximately 0.4 mg/g of larvae or 0.2 mg/larvae. The rVP2H particles have a uniform morphology of dodecahedral structure with a size of 23 nm in diameter, and the particles could be affinity-purified in one step with immobilized metal-ion affinity chromatography (IMAC) from the larvae homogenate. The recovery of rVP2H protein was approximately 55% following IMAC and the protein was obtained with a purity of around 90%. An additional purification step of ammonium sulphate precipitation was added to speed up the process of microfiltration and ultrafiltration of the homogenate prior to IMAC. This step enhanced the final purity of rVP2H protein to 99%, demonstrating that the purification protocol developed herein was a powerful strategy for obtaining highly pure rVP2H protein from insect larvae. The immunogenicity and protective properties of the larvae-derived rVP2H protein were evaluated using a chicken protection assay. When larvae-derived rVP2H protein was intramuscularly injected into specific-pathogen-free chickens (20 μg/bird), high titres of virus-neutralizing antibodies were induced and the chickens were protected from the infection of a very virulent strain of IBDV isolated locally.     

Chimeric infectious bursal disease virus-like particles expressed in insect cells and purified by immobilized metal affinity chromatography.

Chimeric virus-like particles (VLPs) of infectious bursal disease virus (IBDV) were produced by coinfecting Spodoptera frugiperda (Sf-9) insect cells with two recombinant baculoviruses, vIBD-7 and vEDLH-22. vIBD-7 encodes VP2, VP3, and VP4 of the IBDV structural proteins. vEDLH-22 encodes VP2 with five histidine residues at the carboxy-terminus (VP2H). Coinfection produced hybrid VLPs composed of VP2, VP2H, and VP3. The additional histidine residues on VP2H enabled the efficient purification of VLPs based on immobilized metal affinity chromatography (IMAC). These results demonstrated that the VLPs formed are comprised of chimeric subunits with attached affinity ligands, and further, that sufficient His5 ligand was available for binding to the IMAC metal-chelating resin. Additionally, these novel particles were fully characterized for antigenicity by a series of monoclonal antibodies, and appeared identical to the two wild-type IBDV strains contributing subunits to the chimeric VLP. IMAC purification provides a promising low-cost and simple scheme to purify VLPs as vaccines.     

传染性法氏囊病病毒AH1株vp2基因在昆虫细胞中的表达与应用

将近期引起传染性法氏囊病(IBD)免疫预防失败的传染性法氏囊病病毒(IBDV)vp2基因,定向克隆入杆状病毒表达系统的供体质粒pFastBacHTA中,构建重组供体质粒pFastBacHTA-VP2,转化Escherichia coli DH10Bac感受态,筛选重组杆状病毒表达质粒pBac-VP2。用pBac-VP2转染Sf9昆虫细胞,获得重组杆状病毒vBac-VP2。对重组杆状病毒vBac-VP2感染的Sf9细胞,用间接免疫荧光试验(IFA)检测,具有特异性荧光;用IBDV抗体夹心ELISA检测,呈阳性反应,抗原效价达到1.6×103;用Western blotting分析,在53kDa处出现一条特异蛋白条带;电镜观察,重组Vp2蛋白能够自组装成病毒样颗粒,在感染细胞中发现了"包涵体样"结构。用HisTrap HP亲和层析柱纯化的重组Vp2蛋白作为包被抗原,建立的IBDV抗体间接ELISA检测方法具有良好的特异性。用重组杆状病毒感染的Sf9昆虫细胞裂解物,免疫2周龄SPF鸡,一次免疫14d后,ELISA检测抗体效价为8×102,中和抗体效价为1106,攻毒实验的存活率为30%;二次免疫14d后,ELISA抗体效价为3.2×103,中和抗体效价为2536,存活率为100%。在实验观察7d内,重组Vp2蛋白免疫保护鸡未显任何临床症状和病理变化,法氏囊/体重比高于对照组(P0.05)。本实验制备的病毒样颗粒重组Vp2蛋白在研制新型IBD基因工程疫苗和检测试剂方面显示出了应用前景。     

Expression of immunogenic VP2 protein of infectious bursal disease virus in Arabidopsis thaliana

VP2 protein is the major host-protective immunogen of infectious bursal disease virus (IBDV) of chickens. Transgenic lines of Arabidopsis thaliana expressing recombinant VP2 were developed. The VP2 gene of an IBDV antigenic variant E strain was isolated, amplified by RT-PCR and introduced into a plant expression vector, pE1857, having a strong promoter for plant expression. A resulting construct with a Bar gene cassette for bialaphos selection in plant (rpE-VP2) was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-VP2 construct was used to transform Ar. thaliana and transgenic plants were selected using bialaphos. The presence of VP2 transgene in plants was confirmed by PCR and Southern blot analysis and its expression was confirmed by RT-PCR. Western blot analysis and antigen-capture ELISA assay using monoclonal anti-VP2 were used to determine the expression of VP2 protein in transgenic plants. The level of VP2 protein in the leaf extracts of selected transgenic plants varied from 0.5% to 4.8% of the total soluble protein. Recombinant VP2 protein produced in plants induced antibody response against IBDV in orally-fed chickens.     

In vitro assembly of chimeric virus-like particles composed of a porcine circovirus 2b capsid protein and a B-cell epitope of infectious bursal disease virus

Biotechnology Letters - To develop a method for in vitro assembly of recombinant proteins expressed in E. coli into chimeric virus-like particles (cVLPs). A fusion protein (Bepi-Cap-A) between...     

One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purity, as determined by SDS-PAGE analysis, was greater than 80%. The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca(2+) was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.     

A modified metal-ion affinity chromatography procedure for the purification of histidine-tagged recombinant proteins expressed in Drosophila S2 cells

We have developed a modified method of immobilized metal-ion affinity chromatography (IMAC) that can be used for the purification of histidine-tagged proteins from conditioned medium containing free copper ions. Classical methods of IMAC purification, using resins such as Ni-NTA, have proven inefficient for this type of purification and require multiple steps due to the interference of divalent copper ions with the binding of His-tagged protein to the charged resin. In contrast, this modified IMAC procedure, using chelating Sepharose instead of Ni-NTA, enables efficient purification from copper-containing medium in a single step. This method appears to rely upon a preferential interaction of protein-copper complexes with immobilized chelating resin. We have utilized this method to purify active, His-tagged murine interleukin 12 from the conditioned medium of Drosophila S2 cells coexpressing recombinant p40 and His-tagged p35 subunits and for the purification of the extracellular domain of the erythropoietin receptor. This method should be applicable to the purification of a wide variety of His-tagged fusion proteins expressed in Drosophila cells and in other systems where free metal ions are present.     

C terminus of infectious bursal disease virus major capsid protein VP2 is involved in definition of the T number for capsid assembly

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.     

Overexpression of recombinant infectious bursal disease virus (IBDV) capsid protein VP2 in the middle silk gland of transgenic silkworm

Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious disease affecting young chickens and causes serious economic losses to the poultry industry worldwide. Development of subunit vaccine using its major caspid protein, VP2, is one of the promising strategies to protect against IBDV. This study aim to test the feasibility of using silkworm to produce recombinant VP2 protein (rVP2) derived from a very virulent strain of IBDV (vvIBDV). A total of 16 transgenic silkworm lines harboring a codon-optimized VP2 gene driven by the sericin1 promoter were generated and analyzed. The results showed that the rVP2 was synthesized in the middle silk gland of all lines and secreted into their cocoons. The content of rVP2 in the cocoon of each line was ranged from 0.07 to 16.10 % of the total soluble proteins. The rVP2 was purified from 30 g cocoon powders with a yield of 3.33 mg and a purity >90 %. Further analysis indicated that the rVP2 was able to tolerate high temperatures up to 80 °C, and exhibited specific immunogenic activity in mice. To our knowledge, this is the first report of overexpressing rVP2 in the middle silk gland of transgenic silkworm, which demonstrates the capability of silkworm as an efficient tool to produce recombinant immunogens for use in new vaccines against animal diseases.     

Identification and molecular characterization of the RNA polymerase-binding motif of infectious bursal disease virus inner capsid protein VP3

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most important infectious poultry diseases. Major aspects of the molecular biology of IBDV, such as assembly and replication, are as yet poorly understood. We have previously shown that encapsidation of the putative virus-encoded RNA-dependent RNA polymerase VP1 is mediated by its interaction with the inner capsid protein VP3. Here, we report the characterization of the VP1-VP3 interaction. RNase A treatment of VP1- and VP3-containing extracts does not affect the formation of VP1-VP3 complexes, indicating that formation of the complex requires the establishment of protein-protein interactions. The use of a set of VP3 deletion mutants allowed the mapping of the VP1 binding motif of VP3 within a highly charged 16-amino-acid stretch on the C terminus of VP3. This region of VP3 is sufficient to confer VP1 binding activity when fused to an unrelated protein. Furthermore, a peptide corresponding to the VP1 binding region of VP3 specifically inhibits the formation of VP1-VP3 complexes. The presence of Trojan peptides containing the VP1 binding motif in IBDV-infected cells specifically reduces infective virus production, thus showing that formation of VP1-VP3 complexes plays a critical role in IBDV replication.     

The 2.6-Angstrom structure of infectious bursal disease virus-derived T=1 particles reveals new stabilizing elements of the virus capsid

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T=1 subviral particles (SVP) when individually expressed as a chimeric gene. We have determined the crystal structure of the T=1 SVP to 2.60 A resolution. Our results show that the 20 trimeric VP2 clusters forming the T=1 shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T=1 particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions.     

The oligomerization domain of VP3, the scaffolding protein of infectious bursal disease virus,plays a critical role in capsid assembly

Infectious bursal disease virus (IBDV) capsids are formed by a single protein layer containing three polypeptides, pVP2, VP2, and VP3. Here, we show that the VP3 protein synthesized in insect cells, either after expression of the complete polyprotein or from a VP3 gene construct, is proteolytically degraded, leading to the accumulation of product lacking the 13 C-terminal residues. This finding led to identification of the VP3 oligomerization domain within a 24-amino-acid stretch near the C-terminal end of the polypeptide, partially overlapping the VP1 binding domain. Inactivation of the VP3 oligomerization domain, by either proteolysis or deletion of the polyprotein gene, abolishes viruslike particle formation. Formation of VP3-VP1 complexes in cells infected with a dual recombinant baculovirus simultaneously expressing the polyprotein and VP1 prevented VP3 proteolysis and led to efficient virus-like particle formation in insect cells.     

The capsid of infectious bursal disease virus contains several small peptides arising from the maturation process of pVP2

The capsid proteins VP2 and VP3 of infectious bursal disease virus, a birnavirus, are derived from the processing of a large polyprotein: NH2-pVP2-VP4-VP3-COOH. Although the primary cleavage sites at the pVP2-VP4 and VP4-VP3 junctions have been identified, the proteolytic cascade involved in the processing of this polyprotein is not yet fully understood, particularly the maturation of pVP2. By using different approaches, we showed that the processing of pVP2 (residues 1 to 512) generated VP2 and four small peptides (residues 442 to 487, 488 to 494, 495 to 501, and 502 to 512). We also showed that in addition to VP2, at least three of these peptides (residues 442 to 487, 488 to 494, and 502 to 512) were associated with the viral particles. The importance of the small peptides in the virus cycle was assessed by reverse genetics. Our results showed that the mutants lacking the two smaller peptides were viable, although the virus growth was affected. In contrast, deletions of the domain 442 to 487 or 502 to 512 did not allow virus recovery. Several amino acids of the peptide 502 to 512 appeared essential for virus viability. Substitutions of the P1 and/or P1" position were engineered at each of the cleavage sites (P1-P1": 441-442, 487-488, 494-495, 501-502, and 512-513). Most substitutions at the pVP2-VP4 junction (512-513) and at the final VP2 maturation cleavage site (441-442) were lethal. Mutations of intermediate cleavage sites (487-488, 494-495, and 501-502) led to viable viruses showing different but efficient pVP2 processing. Our data suggested that while peptides 488 to 494 and 495 to 501 play an accessory role, peptides 442 to 487 and 502 to 512 have an unknown but important function within the virus cycle.     

Stable synthesis of viral protein 2 of infectious bursal disease virus in Saccharomyces cerevisiae.

Viral protein 2 (VP2) from infectious bursal disease virus and its precursor polyprotein (N-VP2-VP4-VP3-C), in the absence of their native N-terminal region (19 amino acids), required fusion of yeast presequences for their stable synthesis in Saccharomyces cerevisiae [Jagadish et al., Gene 95 (1990) 179-186]. Restoration of the missing 19 aa resulted in stable synthesis of VP2, indicating the significance of the N-terminal region in protein stability.     

Immunofluorescent detection of double-stranded RNA in cells infected with reovirus, infectious pancreatic necrosis virus, and infectious bursal disease virus

Urea treatment of ethanol-fixed virus-infected cells exposed nucleic acid antigens for immunofluorescence. Three double-stranded (ds) RNA-containing viruses showed bright fluorescence using antibodies against dsRNA. Three single-stranded RNA-containing viruses showed less intense fluorescence with anti-dsRNA. Four out of five cell lines persistently infected with various RNA-containing viruses showed no dsRNA detectable by immunofluorescence.     

The major antigenic protein of infectious bursal disease virus, VP2, is an apoptotic inducer.

Infectious bursal disease virus (IBDV) is the causative agent of an economically important poultry disease. Vaccinia virus recombinants expressing the IBDV mature structural capsid proteins VP2 and VP3 were generated by using vectors for inducible gene expression. Characterization of these recombinant viruses demonstrated that expression of VP2 leads to induction of apoptosis in a variety of mammalian cell lines. Transfection of cell cultures with a expression vector containing the VP2 coding region under the control of the immediate-early promoter-enhancer region of human cytomegalovirus also triggers programmed cell death. The apoptotic effect of VP2 is efficiently counteracted by coexpression of the proto-oncogene bcl-2. The results presented demonstrate that VP2 is a bona fide apoptotic inducer. Evaluation of the significance of this finding for the virus life cycle must await further research.     

Vaccine development through terminal deletions of an infectious bursal disease virus protein 2 precursor variant

VP2 is the primary host-protective immunogen of infectious bursal disease virus (IBDV), the agent that causes the highly contiguous infectious bursal disease (IBD). Previous studies have shown that a C-terminal his-tagged 452 amino acid residue VP2 precursor variant (VP2-452H) can form an immunogenic subviral particle (SVP). A set of his-tagged N- and C-terminal VP2-452 deleting mutants (designated as N5-452H, N10-452H, N20-452H, N40-452H, VP2-441H, VP2-437H, VP2-411H and VP2-399H) was expressed in insect cells to discover the role of both N- and C-termini on the assembly of SVP and to develop an efficient SVP-based vaccine. Among these mutants, the expression level of N5-452H was the highest. Results of ultracentrifugation and electron microscopy also indicated that mutants of N-terminal deletion N10-452H, N20-452H and N40-452H or C-terminal deletion VP2-411H and VP2-399H lost the capability to self-assemble SVP. The other mutants, N5-452H, VP2-441H and VP2-437H, formed SVP. Additionally, SVP formed by N5-452H could not only be single-step purified by immobilized metal-ion affinity chromatography (IMAC), but it could also induce a high titer of neutralizing activity to protect chicks from the infection of IBDV at a low dosage (0.2 μg), suggesting that SVP formed by N5-452H can be an alternative vaccine candidate for the prevention of IBD.     

Rapid purification of calmodulin and S-100 protein by affinity chromatography with melittin immobilized to sepharose

Melittin-Sepharose was prepared for Ca2+-dependent affinity chromatography of calmodulin and S-100 protein. This matrix exhibits extremely high capacity (approximately 10 mg calmodulin/ml gel), low nonspecific binding, and excellent recovery (greater than 90%) under optimal conditions. Recovery of calmodulin from melittin-Sepharose was related to the degree of saturation of column capacity with lower yields when only partial saturation was achieved. Large-scale, simultaneous purification of calmodulin and S-100 protein from brain was carried out using selective adsorption to organomercurial agarose followed by melittin-Sepharose chromatography; yields were 250-300 mg of calmodulin and 200-300 mg of S-100 per kg tissue. Calmodulin also was purified in a single step from bovine testis supernatant using melittin-Sepharose in yields comparable to those from brain.     

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.     

Recombinant canarypox virus expressing the VP2 protein of infectious bursal disease virus induces protection in vaccinated SPF chickens

正Dear Editor,Infectious bursal disease virus(IBDV)causes infectious bursal disease,a highly contagious immunosuppressive disease that affects young chickens and causes economic losses in the poultry industry worldwide.IBDV replicates mainly in actively dividing B lymphocytes within the bursa of Fabricius(BF),leading to immunosuppression in affected flocks(Mahgoub et al.,2012).Viral protein 2(VP2),the only structural component of the IBDV