The baculovirus expression vector system (BEVS) is an effective and widely used method for the production of recombinant proteins in insect cells or larvae. However, the expression efficiency of foreign proteins using the polyhedrin promoter could not obtain the protein yields observed for native polyhedrin. To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Among the fusion-expressed protein in nucleus and cytoplasm, the most hyper-expression was observed in the fusion of amino acids 19 to 110 and 32 to 59 of polyhedrin. Additionally, the several proteins expressed by the partial polyhedrin-fused expression system was markedly increased. However, we identified that hyper-expression of target protein varied depending on the partial polyhedrin. Therefore, we constructed the virus inducible partial polyhedrin fusion transient expression system. This system amenable for screening of suitable partial polyhedrin to produce the target protein. The present study suggests a new option for higher expression of useful foreign recombinant protein using the partial polyhedrin fusion expression in baculovirus.

Porcine parvovirus (PPV), a member of the genus Parvovirus, family Parvoviridae, is a significant causative agent in porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a non-enveloped virus and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2 is the main target for neutralizing antibodies in PPV. When VP2 was expressed in large amounts, it assembled into virus-like particles (VLPs) similar in size and morphology to the original virions. In this study, we generated the recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) to express the VP2 protein. Expression of the VP2 protein was analyzed by SDS-PAGE and Western blot. The recombinant VP2 protein of approximately 64 kDa was detected by both analyses. The formation of VLP by recombinant VP2 was confirmed through transmission electron microscopy examination. The purified VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm.

A novel recombinant bacmid, bEasyBac, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBac, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the non-recombinant background. The bEasyBac bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBac was transposed with pDualBac-EGFP, the resulting recombinant virus, AcEasy-EGFP, showed comparable levels of EGFP expression efficiency to the plaque-purified recombinant virus AcEGFP, which was constructed using the bAcGOZA system. In addition, no non-recombinant backgrounds were detected in unpurified AcEasy-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.

To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of polyhedrin fragments were investigated by fusion expression them with the enhanced green fluorescence protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The increase of EGFP production by fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production of EGFP was shown by the mutation of the NLS within the fused polyhedrin fragment. Among the fusion expressed protein in cytoplasm, the most hyper-expression was observed in the fusion of amino acids 32 to 59 of polyhedrin. Polyhedrin fragment fusion expression with classical swine fever virus E2 protein also resulted hyper-enhanced expression of E2 protein. However, the fusion expression of porcine circovirus ORF2 with polyhedrin fragment did not show significant enhance of ORF2 production. These results suggested that the enhancement of foreign protein production when fused with polyhedrin is caused by the enhanced stability of expressed protein.

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to meet a multi-parellel process. We have developed a novel recombinant bacmid, bEasyBm that enabling easy and fast generation of pure recombinant virus without any purification step. In the bEasyBm, attR recombination sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus early promoter. Therefore, only when the barnase gene was replaced to gene of interest, the bEasyBm could replicate in host insect cells. When the bEasyBm was transposed with pDualBac-EGFP and pDualBac-LUC respectively, there were no non-recombinant backgrounds were detected from unpurified BmEasy-EGFP or BmEasy-LUC stocks. In addition, the resulting recombinant virus, BmEasy-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, BmEGFP, which was constructed using bBmGOZA system. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established.

Porcine Circovirus Type2 (PCV2), a single-stranded DNA virus associated with Postweaning multisystemic wasting syndrome(PMWS) of swine, has two major open reading frames, ORF1 and ORF2. The genomic size and molecular weight of ORF2 is respectively 699bp, 28kDa. ORF2 encodes the capsid protein (structural protein) that has type-specific epitopes and is very immunogenic and associated with the induction of neutralizing antibodies, suggesting its potential use in diagnostic assays as well as vaccine development. For efficient production of the capsid proteins, we expressed the PCV2 ORF2 gene with baculovirus in the insect cells. In this study, PCV2 ORF2 was appropriately ligated into the baculovirus transfer vector, pBacPAK9 and pB9-Acpol19-110-EK. Sf21 cells were transfected with a mixture of the purified recombinant transfer vector and bAcGOZA. We generated and purified recombinant viruses containing PCV2 ORF2, and named rAc-B9-PCV2ORF2 and rAc-B9-19-110-EK-PCV2ORF2, respectively. Expression levels of capsid fusion proteins with a partial polyhedrin region of AcNPV more increased than recombinant proteins from non-fusion expressed. Also, expression efficiency increased over time and differed at MOI. As a results, fusion expression of porcine circovirus type2 ORF2 using baculovirus could be utilized as an alternative expression method to produce recombinant antigen against PCV2 infection and is worthy of further investigation.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). To enhance the expression level of baculovirus vector system, we constructed several fusion vectors using various fragments of the polyhedrin. The polyhedrin fragments were genetically fused to the enhanced green fluorescent protein (eGFP) under the control of polyhedrin promoter, and their expressions were analyzed in Sf21 insect cells. Expression of the fusion protein was identified by SDS-PAGE and Western blot analysis using anti-GFP and anti-Polyhedrin. The expression level of eGFP was markedly increased by the fusion of partial polyhedrin. Also, the fluorescence intensity of fusion proteins was higher than that of non-fusion protein. Confocal laser scanning microscopy demonstrated that fusion proteins were localized to the cytosol or nucleus of insect cells. In additional, the glycoprotein E2 (gE2) of classical swine fever virus (CSFV) expressed by the these vectors was dramatically increased and its immunogenicity was proofed using experimental animal guinea pigs that were immunized with the partial polyhedrin containing gE2. This study provides a new option for the higher expression of useful foreign recombinant protein by using the partial polyhedrin in BEVS.

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus within the family Reoviridae, is the causative agent of maize rough dwarf and rice black-streaked dwarf diseases, both of which can lead to severe yield losses in east Asia. Although molecular approaches such as RT-PCR have potential for detection and diagnosis of this virus infections, their impact on high throughput certification is still limited. Therefore, the development of an antibody-based assay for rapid and effective diagnosis of RBSDV is preferable. In this study, we collected RBSDV from rice with rough dwarf disease and its complete nucleotide sequences of 10 genomic segments encoding 12 non-overlapping ORFs were determined. Among 12 ORFs, ORF1, 2 and 12 showed high level of similarities with the RdRp, major core protein and major outer shell protein, respectively. These ORFs were expressed as polyhedrin fusion protein or full-length soluble protein using baculovirus expression system for the preparation of specific antibody against RBSDV, which could be useful for the detection and diagnosis of this virus.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. The objective of this study was to enhance production of E2 protein by fusion with partial polyhedrin of nucleopolyhedrovirus in insect cells. We generated various E2 form by fusion with different combinations of the partial polyhedrin and deletion of the C-terminal transmembrane region (TMR). Expression of the E2 protein was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. The fusion expression of an E2 protein with the partial polyhedrin markedly increased expression levels. Also, expression of E2 proteinlacking TMR region was higher than that of intact E2 protein. As a result, the fusion expression of E2 protein lacking the C-terminal TMR with partial polyhedrin was significantly increased in insect cells. These suggest that the fusion of target foreign protein with partial polyhedrin could enhance significantly the production of target protein.

Porcine circovirus type 2 (PCV2) is associated with porcine circovirus diseases (PCVD), of which postweaning multisystemic wasting syndrome is considered to cause considerable economic losses in pig industry worldwide. As the virus-like particle (VLP) is a highly effective type of subunit vaccine and has unique advantages in terms of safety and immunogenicity, this study aimed to develop PCV2-like particles, which matched currently circulating Korean PCV2 and were applicable as vaccines. The ORF2 genes encoding PCV2 capsid protein were amplified from the PCV2 subgroup 1A/B Korean C275 isolate and the subgroup 2E C94 isolate by PCR assay with primer pair specific to PCV2 ORF2 gene, and were cloned into baculovirus transfer vector. Recombinant baculovirus was generated by cotransfection with the transfer vector and linear baculovirus DNA into the Sf9 cells, and then by plaque purification. Expression of PCV2 capsid protein was determined by the indirect immunofluorescence and Western blotting assays, and electron microscopy. By both immunological assays, PCV2 capsid antigen was detected in the Sf9 cells infected with the recombinant baculoviruses. The formation of empty virus particles, characteristic of VLP, was detected in the lysate of Sf9 cells infected with the recombinant baculoviruses by negative electron microscopy. From these results, VLPs of two genogroups of PCV2 were successfully expressed and generated in a baculovirus expression system. It is expected that the expressed VLPs of two genotypic groups can be used for control of PCV2 infection as good vaccine candidates.

Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures and these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, and mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with Polh gene at the N-terminus including an adaptor and enterokinase (EK) site between Polh and EGFP was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells according to three steps; cell harvest, sonication and EK digestion. Through the final enterokinase digestion, EGFP was presented mainly in the supernatant (93.3%) and the supernatant also showed a pure EGFP band. These results suggest that the combined procedure of Polh fusion expression and enterokinase digestion can used for the rapid and easy purification of other proteins.

The flavin-containing monooxygenases (FMOs) (EC 1.14.13.8) are NADPH-dependent flavoenzymes that catalyze oxidation of soft nucleophilic heteroatom centers in a range of structurally diverse compounds including foods, drugs, pesticides, and other xenobiotics. In humans, FMO1 appears to be the predominant form expressed in human fetal liver. cDNA-expressed human FMO and human liver microsomal FMO have been observed to N- and S-oxygenate nucleophilic nitrogen- and sulfur-containing drugs and chemicals, respectively. In the present study, FMOl can be expressed in the baculovirus expression vector system at level of 2.68 nmol FMO1/㎎ of membrane protein. This isoform was examined for its capacity to metabolize methimazole to its S-oxide using thiocholine assay. Kinetic studies of its S-oxide by recombinant human FMO1 result in K_m of 7.66 μM and V_(max) of 17.79 nmol/min/㎎ protein.