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.

Virus-like particles (VLPs) are similar to pathogenic viruses, but because they have no nucleic acid, they have excellent safety and immunogenicity and are used as a good vaccine material. However, in the selection of various structural proteins of pathogenic viruses to form VLPs, all expression systems consume a lot of time in common. Among them, the baculovirus expression system causes additional time consumption to construct the recombinant baculovirus. Therefore, there is a need for a system that can rapidly determine the structural proteins required for effective VLP production. This study aims at solving this problem by constructing a BmNPV inducible expression platform through the construction of vectors induced by BmNPV. The platform was evaluated for overexpression using EGFP. We also confirmed the formation of virus-like particles through overexpression of canine parvovirus structural proteins.

The virus-like particle (VLP) is similar to a pathogenic virus and has a high immunogenicity. However, in the selection of various structural proteins to form VLP, all expression systems commonly consume most of the time and suffer from various difficulties. Therefore, there is a need for a system that can rapidly determine the structural proteins required for effective VLP production. This study aims to construct a transient expression platform using insect cells to solve this problem. Plasmid-based expression vectors and baculovirus-inducible expression vectors were constructed. The vectors were evaluated for overexpression using EGFP. We also confirmed the formation of virus like particles through overexpression of virus structural proteins.

The population of managed honeybees has been dramatically declining the recent past in worldwide. N. ceranae causessignificant detriment to honey production and results in economic losses critically. In our knowledge, Fumagillin is theonly antibiotic approved for control of nosemosis in honeybees. In this study, to select isolate with anti-Nosema activityagainst N. ceranae. Entomopathogenic fungi cultural filtrates were screened using in vitro polar tube germination assay.These fungi cultural filtrates were used to evaluate the safety of honeybees and their inhibition of nosemosis. As a result,P. marquandii 364 and Pochonia sp. 60 showed inhibitory activity on the growth of N. ceranae in honeybees and didnot significantly affect the survival rate of honeybees. These may be employed as antibiotic agents and a good featureto be used in the development of new biocontrol agents of nosemosis.

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.

The two-spotted spider mite, Tetranychus urticae, and gray mold disease caused by Botrytis cinerea, are importantpest and plant disease. To control these, many people have been relied on chemical methods for a long time. However,their continuous use has resulted in resistance of pest and disease. To surmount or avoid these problems, we evaluatedantifungal activity of the selected fungi with high virulence to mite to explore the potential for the dual control of notonly T. urticae but also B. cinerea. To maximize the use of spores and cultural filtrates, the virulence to mite was evaluatedusing culture products, and effective culture condition was investigated against blastospore production and antifungal activity.Consequently, the 2 fungal isolate selected in this study were confirmed to have diverse potential and would be usedeffectively for dual control agents against the two-spotted spider mite and plant diseases

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.

The green peach aphid, Myzus persicae, is one of the most important pests of vegetable crops worldwide. This species cause direct damage by feeding on plant nutrients and indirect damage as transmits many virus vectors. The control of aphid has relied on the use of chemical insecticides and their intensive use over many years has led to the development of resistance. To surmount or avoid these problems, we screened the entomopathogenic fungi against green peach aphid and evaluated their virulences. Several entomopathogenic fungal isolates were selected with high virulence to green peach aphid. Additionally, the antimicrobial activity of the selected fungal isolates was tested and analyzed to phytopathogens. Consequently, these entomopathogenic fungi would be used effectively for dual control agents for the green peach aphid and phytopathogen.

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.

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.