The baculovirus-insect cell expression system has been widely used method for the recombinant protein expression. The present study has several limitation. In this study, we constructed vectors consisting of transcriptional enhanced factor and promoter that improve the expression level. To confirm the usefulness of these vector system, Human papillomavirus (HPV) VLPs have been expressed by baculovirus hyper expression system. HPV VLPs were purified using a CaptoTM Core 700 (GE Healthcare Life Sciences) chromatography approach. Baculovirus hyper expression system production efficiency was influenced by the HPV VLPs production. HPV VLPs vaccination to BALB/c mice induced the generation of antibody confirmed by ELISA. This study could provide improvements on the vaccine production for the development of VLP vaccines high expression of useful heterologous proteins.

The baculovirus expression system is a very useful tool widely used for expression of foreign proteins. To use the baculovirus expression system, a recombinant baculovirus must be prepared. The development of the Bac to Bac system has reduced the time and effort required to produce recombinant baculovirus. But, it will take at least two weeks. Further, it takes more time to measure the activity of recombinant baculovirus. In order to overcome this problem, a virus inducible expression system is being studied recently. Although baculovirus is able to rapidly express foreign proteins, it still has a low expression level. Thus, in this study, we aimed to construct a novel baculovirus inducible expression vector that not only shortens the production time of protein but also can express at a high level. The novel baculovirus inducible expression vector has been evaluated using EGFP and is expected to be a very useful tool for production of various proteins.

Among the various expression systems used for foreign protein expression, baculovirus expression system (BES) has the high level of post-translational modification ability such as glycosylation, folding and disulfide bonding. BES is widely used now in the production of VLPs because it is possible for the efficient multi-gene expression. However, there are not many cases of VLPs being manufactured through BES. Therefore, in this study, three improvements were made to increase the productivity of VLP through BES. A new heper enhanced expression vector was constructed to increase the expression of structural proteins of virus-like particles, and baculovirus bacmid was modified to increase production time. In addition, an easy purification system was constructed to efficiently purify VLP, and finally the construction of BES optimized for VLP production was completed.

The baculovirus expression vector system (BEVS) is an effective and widely used system for the production of recombinantproteins in insect cells or larvae. However, the expression efficiency of recombinant proteins using the polyhedrin promotercould not acquire the protein yields observed for native polyhedrin. In this study, we tried to develop hyper expressionvector by the optimal combination of previously reported various enhancer factors. The selected enhancer factors for optimalexpression consists homologous region5 (hr5), VP39 promoter and burst sequences. Seven recombinant viruses were madeto compare EGFP expression level. Each recombinant viruses showed different expression levels respectively, and themost of expression level was observed with higher than those of the previous vectors. This study suggests a new optionfor hyper expression of useful recombinant protein using the BEVS.

Viral particles of Porcine epidemic diarrhea virus (PEDV) consist of a four structural proteins. Among them Spikeprotein mediated responsible for receptor binding and membrane fusion during viral infection and therefore the main targetof neutralizing antibodies. Virus-like particles (VLPs) are consisted of one or more viral structural proteins, and theirmorphologies closely resemble those of the native virus. VLPs have no virulence and can elicit robust immune responsesas compared with inactivated or live-attenuated virus vaccines. Thus, in this study, we tried two methods for VLP constructionin Bombyx mori, one is traditional method and the other is chimeric VLP method using the influenza matrix protein.Both methods could produce successfully PEDV VLPs.

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 enhancer factor were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). As a hyper expression factor, the optimal hyper BEVS was constructed by combining Hr5 sequence, VP39 promoter and Burst sequences. Additionally, the proteins expressed by the hyper expression system was markedly increased. This study suggests a new option for higher expression of useful foreign recombinant protein using the BEVS.

Recombinant proteins including a polypeptide fusion partner, termed affinity tag, to facilitate the purification of the target polypeptides are widely used. However, the affinity tag must be removed from the target after the purification process. Recently, the Synechocystis sp. PCC6803 DnaB mini-intein (Ssp DnaB mini-intein) is widely used in Escherichia coli expression systems as the solution of this problem. The Ssp DnaB mini-intein can be induced simply by shifting of pH and temperature, offering a benefit to cleave a peptide bond without using a protease or chemical reagent. Although the utility of this novel tag is widely studied in E. coli, there is no report yet in baculovirus expression vector system (BEVS). In this study, we generated several recombinant baculoviruses to express foreign proteins with Ssp DnaB mini-intein. In conclusion Ssp DnaB mini-intein was good tag also in BEVS with more advantages.

Canine parvovirus (CPV), a member of the genus Parvovirus, family Parvoviridae, is a significant causative agent in Canine reproductive failure, causing serious economic losses in the pet industry. The major capsid protein, VP2 is the main target protein for neutralizing antibodies in CPV. When VP2 was expressed using baculovirus, it was produced abundantly and assembled into virus-like particles (VLPs) similar in size and morphology to the original virions. It was named as CPV-VLP. Additionally, p35 sequences of canine distemper virus (CDV) as T-helper epitope were fusion-expressed with each of N-term, C-term or both sides of CPV-VP2. Production of double antigenic recombinant protein and formation of VLPs were analyzed by SDS-PAGE and transmission electron microscopy, respectively.

Recombinant baculovirus genome that absence part of ORF1629 has used to enhance efficiency of recombinant virus selection because a role of ORF1629 is believed to essential for viral replication in insect cells. It can be recovered by recombination with a transfer vector containing a complete ORF1629. Some recombinant bacmids were generated for this purpose. Recombinant bacmid GOZA, one of them, has a mini-F replicon for Escherichia coli and the partial ORF1629 gene. By accident, we could observe the replication of ApGOZA singly in Sf9 cells. Produced virus from ApGOZA was investigated for the existence of truncated ORF1629 not intact ORF1629 by PCR amplification and genomic sequencing. Also, we could observe the replication of AcGOZA alone in Sf9 cells. To analyze the influence of truncated ORF1629, the viral growth, BV production and polyhedral formation were conducted comparing to wild type virus and recombinat virus containing intact ORF1629. The results suggested the probability that ORF1629 is not essential for replication.

Polydnavirus (PDV) is a group of double-stranded DNA insect viruses. PDV is mutualistic with some ichneumonid and braconid wasps to parasitize specific lepidopteran hosts. The viral genome is located on the wasp chromosome(s) as a proviral form and replicates only at the female reproductive organ during late pupal stage. The viral particles are accumulated in the oviduct lumen and delivered to the parasitized host along with wasp eggs during parasitization. The viral particles enter target tissues in the parasitized larvae and alter host physiological processes for the wasp development by suppressing immune responses and extending larval period. Cotesia plutellae bracovirus (CpBV) is a PDV symbiotic to C. plutellae parasitizing young larvae of the diamondback moth, Plutella xylostella. The viral particles of CpBV encode 157 open reading frames classified into different gene families. CpBV-PTP family is the largest and comprises of at least 40 gene members. CpBV-BEN, CpBV-ELP, and CpBV-IkB families also share common motifs in each gene group. In addition, two homologous genes of CpBV15α and CpBV15β are encoded in a viral genome segment. To apply these viral genes to enhance an alpha-baculovirus in insecticidal activity, they were recombined with AcNPV under a PDV promoter. As a control, under the same promoter, the recombinant baculovirus expressed an enhanced green fluorescence protein (EGFP). Upon injection or oral feeding tests, three different recombinant baculoviruses (AcNPV-ELP1, AcNPV-CpBV15α, AcNPPV-CpBV15β) enhanced the insecticidal activity compared to a control recombinant (AcNPV-EGFP). However, there was a variation in the up-regulation of the insecticidal activities among the recombinants. AcNPV-ELP1 showed the greatest potency in the insecticidal activity against the beet armyworm, Spodoptera exigua, larvae. AcNPV-ELP1 exhibited a significant variation in insecticidal activity among different larval stages of S. exigua. In the fifth instar, 1.435x107 PIB treatment of AcNPV-ELP1 showed a median lethal time at 112.7 h. ELP1 protein was detected in the hemolymph at 24 h after the viral treatment. Foliar spray of AcNPV-ELP1 was performed in pot assay and resulted in 88% control efficacy against S. exigua, while control efficacies of AcNPV-EGFP and bifenthrin (a pyrethroid insecticide) resulted in 65% and 96%, respectively. These results suggest that a PDV gene, ELP1, may be applied to develop a novel control agent by ameliorating commercial microbial insecticides or by generating transgenic crops.

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.

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of the NeuroBactrus, the Bacillus thuringiensis cry1-5 crystal protein gene was introduced into the Autographa californica nucleopolyhedrovirus(AcMNPV) genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an approximately 65-kDa active toxin. In addition, qPCR revealed that the neurotoxin was expressed from the early phase of infection. The NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time(LT50) against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Re-recombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins(Bt toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passed NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that the NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.

The antigen GA733 is a cell-surface highly expressed glycoprotein on most human colorectal carcinomas. GA733 can be characterized as a cancer vaccine. In this study, GA733 was fused to the human immunoglobulin IgG Fc fragment to become recombinant gene GA733-Fc. Based on this, 4 recombinant genes were constructed as follows: GA733-Fc with signal peptide sequence and fusion of ER retention sequence (KDEL) (spGA733-FcK), GA733-Fc with signal sequence (spGA733-Fc), GA733-Fc fused to ER retention sequence (GA733-FcK) without signal peptide and GA733-Fc without signal peptide. Baculovirus-insect cell expression system is widely used for the high level production of recombinant proteins especially for glycoproteins. Constructed 4 recombinant genes were cloned to baculovirus express vectors. DH10Bac E.coli.-mediated transformation was used to generate recombinant bacmid DNA. Recombinant DNA was confirmed by PCR. Insect cell was transfected by bacmid to produce the recombinant baculovirus infects insect cell to produce recombinant protein. Western blot and sandwich ELISA showed the expression of recombinant proteins. Each cell lines (sf9 and HighFive) differed in recombinant proteins production level and protein secretion capability. N-Glycosylation analysis showed the function of signal peptide and ER retention sequence (KDEL). Taken together, baculovirus-insect cell system can be used to express recombinant GA733-Fc glycoproteins.

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.

Although baculoviruses have a long history of safe use as specific, environmentally benign insect control agents, their use has been limited by several factors, especially their slow speed of action. In this study, we intended to improve the insecticidal activities of Autographa californica nucleopolyhedrovirus (AcMNPV) by expressing Kunitz-type toxin isolated from venoms of Bombus ignitus or Araneus ventricosus. For this, recombinant AcMNPVs, AcBi-KTT, AcAv-Tox1 and AcAv-Tox2 expressing Bi-KTT, Av-Tox1 and Av-Tox2, respectively, under the control of p10 gene promoter were constructed. While polyhedra produced by these recombinant viruses were identical to those of the wild-type AcMNPV in shape, their sizes were relatively smaller than those of the AcMNPV. Among recombinant viruses, AcBi-KTT and AcAv-Tox2 showed significant reduction in median lethal time (LT50) against Spodoptera exigua larvae. Especiaaly, these two viruses showed about 6.2~10-folds higher polyhedra production rate compared to that of the AcMNPV. These results suggested that Kunitz-type toxins from insect venom could be successfully applied to improve insecticidal activity of baculoviruses.

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.