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.

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.

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.