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.

A 7-year-old, spayed female, domestic short hair cat showed signs of a 2-week history of chronic anorexia, depression, and severe weight loss. Upon physical examination, pyrexia, mild gingivitis, and pale mucus membranes were noted. Laboratory analysis revealed normocytic normochromic non-regenerative anemia, severe thrombocytopenia, and hypergammaglobulinemia. Serum protein electrophoresis revealed the presence of elevated alpha-2 fraction within the globulin concentration. Based on history, clinical signs, and laboratory results, systemic viral infection was strongly suspected. Reverse transcriptase polymerase chain reaction identified the presence of feline immunodeficiency virus (FIV) in the serum. Furthermore, gene sequencing revealed the virus as FIV subtype A. Treatment with anti-retroviral agents, including azidothymidine (AZT) and recombinant human interferon-alpha, was continued for 4 weeks. However, the patient’s clinical condition deteriorated, resulting in death 1 month after initiation of treatment due to progressive renal failure. Necropsy and histopathology revealed hepatic and renal necrosis with hyper-cellular bone marrow mainly comprised of myeloid precursor cells. This case report is the first to describe phylogenetic subtyping, anti-retroviral combination treatment, and clinical outcomes in an FIV-infected cat in Korea. In addition, this report suggests that treatment should be initiated during the early phase of infection that could be effective for the virus.

Cystatins (CSTs) are reversible and competitive inhibitors of C1A cysteine proteases, corresponding to papain-like cathepsins in plants and animals. A viral CST (CpBV-CST1) was identified from a polydnavirus, Cotesia plutellae bracovirus. Our previous study indicated that overexpression of CpBV-CST1 interfered with immune response and development of Plutella xylostella larvae. This study produced a recombinant CpBV-CST1 protein (rCpBV-CST1) using bacterial expression system to analyze its inhibitory activity against cysteine protease and physiological role in the parasitism of an endoparsitoid wasp, Cotesia plutellae. The open reading frame (ORF) of CpBV-CST1 encodes a polypeptide of 138 amino acids (15 kDa). rCpBV-cystatin protein in BL21 STAR (DE3) competent cells containing a recombinant pGEX4T-3:CpBV-CST1 was overexpressed by 0.5 mM IPTG for 4 h. In biological activity assay, partially purified GST-fused rCpBV-CST1 showed inhibitory activity against papain. It also inhibited larval development of P. xylostella in a dose-dependent manner. These results suggest that CpBV-CST1 plays a role in retardation of larval development of P. xylostella during parasitism.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). 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). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. 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. Also these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. The production of luciferase was approximately two folds increased by fusion expression with polyhedrin amino acids 32 to 59, and its activity was measured using Luminometer. This study suggests a new option for higher expression of useful foreign recombinant protein using the partial polyhedrin fusion expression in baculovirus.

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.

Colorectal cancer is the third most commonly diagnosed cancer in the world, nearly all patients diagnosed with this cancer die from it. Antibodies are glycoprotein molecules, which can efficiently recognize and eliminate specific pathogenic and disease antigens. Antibody researches for the last several decades have demonstrated the potential of therapeutic antibodies to fight cancer. Monoclonal antibody (mAb) CO17-1A recognizes the tumor-associated antigen GA733-2, a cell surface glycoprotein highly expressed in colorectal carcinoma cell, which is applicable for preventing and curing colorectal cancer. We have currently established baculovirus insect cell expression system to produce anti-colorectal cancer mAb CO17-1A. In this study, mAb CO17-1A was expressed in the transgenic insect cell line SWT4, which has humanized glycosylation processing pathway. Immunoblot confirmed that mAb CO17-1A properly expressed in SWT4. mAb CO17-1A was purified using protein G affinity column. In addition, Maldi-TOF verified that the mAb fused to KDEL, ER retention signal had high mannose type of glycan structure whereas the mAb without KDEL had partially humanized glycan structure. These results suggest that the insect cell expression system with the SWT4 possibly can be used as a useful alternative way to produce full-size mAb with humanized glycan structures for cancer immunotherapy.

Phospholipase A2 (PLA2) catalyze the committed step for eicosanoid biosynthesis and releases arachidonic acid (AA), which is oxygenated into eicosanoids that mediate immune responses in insects. Thus, any inhibition of PLA2 activity would lead to a significant immuno suppression due to lack of eicosanoids. Among more than 15 families of PLA2s, group Ⅳ cytosolic PLA2 (cPLA2) has been mainly associated with the production of eicosanoids associated with immune responses. However, no cPLA2 has been reported in all invertebrates including insects. AcPLA2 candidate gene (SecPLA2) has been identified from a hemocyte transcriptome of the beet armyworm, Spodoptera exigua. RNA interference of SecPLA2 expression significantly reduced cellular immune responses of hemocytes. When the SecPLA2 was expressed and purified, the recombinant SecPLA2 catalyzed a substrate, phosphoatidyl choline, atsn-2 position. Its catalytic activity was sensitive to pH, temperature, and calciumlevel. Furthermore, there combinant SecPLA2 was specifically sensitive to a cPLA2-specificinhibitor, methyl arachidonyl fluorophosphonate.

Chromatin remodelers that include histone methyl transferases (HMTases) are becoming a focal point in cancer drug development. The NSD family of three HMTases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L are bona fide oncogenes found aberrantly expressed in several cancers, suggesting their potential role for novel therapeutic strategies. Several histone modifiers including HMTase have clear roles in human carcinogenesis but the extent of their functions and regulations are not well understood, especially in pathological conditions. The extents of the NSDs biological roles in normal and pathological conditions remain unclear. In particular, the substrate specificity of the NSDs remains unsettled and discrepant data has been reported. NSD2/MMSET is a focal point for therapeutic interventions against multiple myeloma and especially for t(4;14) myeloma, which is associated with a significantly worse prognosis than other biological subgroups. Multiple myeloma is the second most common hematological malignancy in the United States, after non-Hodgkin lymphoma. Herein, as a first step before entering a pipeline for protein x-ray crystallography, we cloned, recombinantly expressed and purified the catalytic SET domain of NSD2. Next, we demonstrated the catalytic activities, in vitro, of the recombinantly expressed NSD2-SET on H3K36 and H4K20, its biological targets at the chromatin.

We prepared the polyclonal antibody anti-20α-hydroxysteroid dehydrogenase (anti-20α-HSD) against the recombinant full-length protein bovine 20α-HSD in Escherichia coli. The specificity of anti-20α-HSD was demonstrated using Chinese hamster ovary (CHO) cells transfected with recombinant bovine 20α-HSD and bovine placental tissues. According to western blot analysis, anti-20α-HSD specifically recognizes the 37-kDa protein bovine 20α-HSD. The protein is not present in untransfected CHO cells. Anti-20α-HSD also recognizes a specific protein in the ovaries and placenta of other animals. Immunostaining was used to detect expression of bovine 20α-HSD protein in the cultured luteal cells during the estrous cycle later.

This study was conducted to investigate the insecticidal capacity of recombinant baculoviruses to Plutella xylostella and Spodoptera exigua larvae. For recombinant viruses, Bacillus thuringiensis cry1-5 crystal protein gene was introduced into the genome by fusion of polyhedrin-cry1-5 under the control of polyhedrin gene promoter. Recombinant AcPolh5-3006BiKTT and AcPolh5-3006 AvTox2 based on BiKTT and AvTox2, respectively, were constructed under the control of early promoter from Cotesia plutellae bracovirus. Mortality of S. exigua larvae was significantly higher when they fed on cabbage coated with ApEGFP (wild type) over 5.0×106 PIBs/ml. For AcPolh5-3006BiKTT and AcPolh5-3006AvTox2, mortality of P. xylostella and S. exigua larvae was significantly higher when they fed on cabbage coated with recombinant baculoviruses over 5.0×106 PIBs/ml and 1.0×104 PIBs/ml, respectively. The value of LD50 was lower in the treatments with AcPolh5-3006BiKTT (P. xylostella:1.2×106, S. exigua:1.3×104) or AcPolh5-3006AvTox2 (P. xylostella:2.3×106, S. exigua:1.4×104) than the treatments with ApEGFP (P. xylostella: not estimated, S. exigua:5.0×105). Survival time (ST50) of P. sylostella larvae was much shorter at AcPolh5-3006BiKTT (29.6h) than AcPolh5-3006AvTox2 (46.2h) while that of S. exigua larvae was much shorter at AcPolh5-3006AvTox2 (95.1h) than AcPolh5-3006BiKTT (101.9h) or ApEGFp (130.7h). The two recombinant baculoviruses were more effective in S. exigua larvae but slower speed of action.

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.

Many thousands of recombinant proteins have been successfully produced in baculovirus - infected insect cells and larvae. In this study, to improve its value and the yield of recombinant protein production, we constructed transgenic silkworm using Heat shock genes with regard to protein folding. This time, we adapted GAL4/UAS system to express at necessary time point and to carry genes for foreign protein. First, we generated two transgenic cells and silkworm lines that carried the silkworm heat shock proteins, UAS-HOP and UAS-HSC70 and UAS-HSP70 and UAS-HSP40 construct plus 3xP3-DsRED. Subsequently, to drive the GAL4 gene as activatorvector, we engineered Baculoviruses that contain the GAL4 under the P10 promoter linked to the expression cassette of interest foreign genes under the polyhedron promoter. Also, activator vector linked to the GAL4 was designed expressing 6xHis and 6xHis–GST tag. Infection of silkworm larvae with recombinant virus, His-tagged human C3d gene was more efficiently produced transgenic silkworm than that of wild-type, but not His-GST tagged. We show the possibilityin use of HSPs transgenic silkworm system by GAL4/ UAS BmNPV that can generate the efficient production of foreign protein.

The silkworm-baculovirus expression system has distinct advantages, such as a high yield and safe usage in vertebrates. Here, we report a novel strategy for the large-scale production of a classical swine fever virus (CSFV) envelope glycoprotein E2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed native polyhedrin and approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg per ml of hemolymph and 0.53 mg per larva at 6 days post-infection. Six-week-old female BALB/c mice that were immunized with the E2ΔC protein purified from solubilized recombinant polyhedraelicited CSFV E2 antibodies, which indicated that the CSFV E2ΔC protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2ΔC protein from recombinant polyhedra contained significant levels of virus neutralization activity. These results demonstrate that the present strategy can be used for the large-scale production of CSFV E2 antigen.

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.

hFSH is a glycoprotein secreted from anterior pituitary and consists of α and β subunits. Because of its major biological functions including sperm formation in the male and for follicular growth, FSH is used to cure woman's sterility. In this study we tried to produce recombinant hFSH in vitro using a retrovirus expression vector. Two major components of the vector we constructed are: (ⅰ) a DNA fragment containing α and β genes fused by a DNA sequence coding carboxyl terminal peptide (CTP) of human chorionic gonadotropin, (ⅱ) a DNA fragment corresponding woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Evaluation of expression profile of the recombinant FSH using reverse transcription PCR and enzyme-linked immunosorbent assay (ELISA). Among three cell lines tested, HeLa cells were the best for hFSH expression (5,395 mIU/ml), then followed by chicken embryonic fibroblast (CEF) cells and Chinese hamster ovary (CHO) cells in the order of hFSH production. In addition to the amount, the FSH produced from HeLa cells was highest in terms of biological activity which was determined by measuring cAMP.