Canine parvovirus (CPV) remains a leading infectious cause of death in canines, especially in young puppies. Though vaccination is being carried out regularly, immunization failures occur, and puppies may be exposed to infection. Virus-like particles (VLPs) act like a subunit vaccine, mimicking the structure of authentic viruses. Therefore, VLPs have the potential to be used as vaccine candidates. Since Viral Protein 2 (VP2), a major structural protein of CPV, is the crucial antigen for CPV, the purpose of this study was to produce a recombinant VP2 of new canine parvovirus-2a using the baculovirus expression system in SF9 insect cells. The results revealed that recombinant VP2 assembles to form VLPs with antigenic properties similar to those of natural CPV, the recombinant VLP can produce a hemagglutination assay (HA) titer (1:210) in SF9 cells. Expression of the recombinant 6-His-tagged VP2 in SF9 cells was confirmed by western blotting. These findings suggest that the recombinant VP2 expressed in this study could be used as an efficient subunit vaccine against CPV infection.
Prolonged communication between oocytes and the surrounding somatic cells is one of the unique reproductive physiology in canine. Paracrine Kit ligand (KITL) signaling is a well-known communication between granulosa cells and the oocyte. KITL is a cytokine growth factor secreted by granulosa cells that signals via the c-kit receptor expressed by oocytes. Paracrine factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), exert their effects by binding with the kinase receptors expressed on the granulosa cells. However, the regulations of GDF9 and BMP15 in the canine KITL expression are currently poorly understood. Therefore, we investigated the effects of GDF9 and BMP15 on the expression of KITL in canine ovarian granulosa cells in vitro.
In Annexin V assay recombinant GDF9 and BMP15 did not induce apoptosis in the cultured ovarian granulosa cells. When treated, FSH significantly increased KITL expression, and hCG suppressed its expression. When both FSH and hCG were treated, the expression of KITL was affected by GDF9 and BMP15 in dose and time dependent manner in the luteal granulosa cells. GDF9 (10 ng/mL) significantly decreased KITL expression after12 h. BMP15 (10 ng/mL) significantly also decreased KITL expression after 24 h. Western blot and immunochemistry results indicate that GDF9 activated Smad2/3. After blocking ALK 4/5/7 receptors by SB, GDF9 failed to activate Smad2/3, also BMP15 did not activate Smad1/5/8 after blocking ALK 2/3/6 receptors by DM. So GDF9 exerts its effects via using ALK 4/5/7 receptors to activate SMAD2/3 signaling, and BMP15 binds ALK 2/3/6 receptors to activate SMAD1/5/8 signaling. The expression of KITL was not changed by SB or DM treatment. However, the effect of GDF9 and BMP15, which decreased the expression of KITL, was suppressed by SB or DM treatment.
In conclusion, this study provides the first evidence that recombinant GDF9 and BMP15 decrease KITL expression in canine ovarian granulosa cells.
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.
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.