Hand, foot, and mouth disease (HFMD) is a highly contagious disease with no specific treatment. Since it is common in immunocompromised children under the age of 5, there is a need to develop a safe vaccine. Virus-like particles (VLPs) are similar structures to viruses with the lack of genetic material which makes them impossible to replicate and infect, and therefore have a high level of biological safety and are considered to have high value as vaccines. In this study, the insect virus expression system that is widely used for vaccine and drug production due to its high post-translational modification efficiency, was used to produce VLPs for Coxsackievirus type A6 and A10, which are recently reported to be the main causes of HFMD. For this purpose, the selection of promoters that can control the timing and intensity of expression of 3CD protein, which is essential for VLPs assembly but has been reported to be cytotoxic, was conducted to construct an optimal expression form for HFMD-VLP.

Viral protein 2 (VP2) of porcine parvovirus (PPV) is responsible for inducing neutralizing antibodies in immunized animals. It is the major viral structural protein. In this study, novel subunit vaccines against PPV based on virus-like particles (VLPs) formed from VP2 proteins (PPV 13-7 Korean strain) were expressed in an insect baculovirus cell system and purified using Ni-NTA affinity column chromatography. These VP2 proteins assembled into virus-like particles (VLPs). They showed antigenic properties similar to those of natural PPV. In addition, they showed high hemagglutination (HA) titers (211 for PPV 13-7 Korean strain). This study provides a foundation for the application of the difference immunization of recombinant protein in the diversity of PPV VP2 genes and in vaccination against PPV in the future.

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.

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.

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.

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.

색소변이체에서 버섯 바이러스의 게놈인 dsRNA가 확인 되었으며, 크기는 각각 5.8kb, 1.8kb 이었다. 느타리바이러스 진단용 프라이머인 PVP로 RT-PCR을 수행한 결과 500bp 크기의 특이밴드가 관찰되었다. 또한 양송이 바이러스 진단용 프라이머 LIVP와 MBVP에서도 특이밴드가 관찰되었으나 양송이 바이러스와는 다른 양상이었다. 원형느타리의 백색변이체 (MGL2205)에 존재하는 바이러스유사입자는 구형이었으며, 크기는 14, 20∼45nm이었다. 균사체의 세포단면을 관찰한 결과 바이러스 순화액에서 확인된 바이러스유사입자와 비슷한 구형의 입자들이 관찰되었으며, 순화된 바이러스와 동일한 입자인지는 추후 확인되어야 할 것으로 사료된다. 원형느타리 백색변이체(MGL2205)에 존재하는 바이러스의 최적 증식 조건은 15℃, pH 6, 배양기간 3주인 것으로 판단되며, 이 결과는 이와 유사한 재배적 조건에서 재확인되어야 할 것으로 사료된다.