Extensive research and testing continue to be conducted for the development of vaccines targeting zoonotic diseases such as brucellosis. In this study, the potential of the DapB as a recombinant protein vaccine to effectively combat Brucella abortus 544 infection in BALB/c mice was evaluated. Western blotting assay results showed that recombinant protein DapB reacted with Brucella-positive serum, indicating its potential immunoreactivity. In vivo results showed that the peripheral blood CD4+ and CD8+ T cell population significantly increased in the DapB-immunized mice group after the first, second and third blood collection, compared to the control group that received PBS. Additionally, at the fourth blood collection, an increase in CD4+ T cell activation was observed in three vaccination groups compared to PBS negative control group. These results indicate the potential of DapB in stimulating cellular immunity. Fourteen days after infection, the bacterial load in the spleen was evaluated. The reduction in bacterial replication in the spleen by both DapB and RB51 highlights their protective efficacy against Brucella infection. These findings contribute to the ongoing efforts in developing effective vaccines against brucellosis and provide valuable insights for further research in this field.

Subunit vaccines are being developed as a potential therapy for preventing microbial pathogen infection. In this study, the immunogenicity of recombinant Brucella (B.) abortus Fe/Mn superoxide dismutase (rFe/Mn SOD) protein as a subunit vaccine against B. abortus was investigated in BALB/c mice model. Brucella Fe/Mn SOD gene was cloned into a pcold-TF DNA vector. The bacterial recombinant protein was expressed using the Escherichia coli DH5α strain with a size of 82.50 kDa. The western blotting assay showed that rFe/Mn SOD reacted with Brucella-positive serum, indicating the potential immunoreactivity of this recombinant protein. After the second and third vaccinations, the peripheral CD4+ T cell population was increased significantly in the rFe/Mn SOD-immunized mice group compared to the PBS control group. Moreover, immunization of this recombinant protein increased the CD4+ T cell population from the first vaccination to the third vaccination. Meanwhile, the CD8+ T cells were slightly enhanced after the second vaccination compared to the first vaccination and compared to control groups. Fourteen days after the bacterial infection, the splenomegaly and the number of bacteria in the spleen were evaluated. The result showed that both rFe/Mn SOD and positive control RB51 decreased the bacterial replication in the spleen and the splenomegaly compared to control groups. Altogether, these results suggested that rFe/Mn SOD could induce host immunity against B. abortus infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans, with a case fatality rate of approximately 35%, thus posing a considerable threat to public health. A lack of approved vaccines or antivirals currently constitutes a barrier for controlling disease outbreaks and spread. In this study, we generated a replication-defective recombinant vesicular stomatitis virus expressing the MERS-CoV spike (S) protein (VSV/MERS). Uncleaved and cleaved S proteins were detected in VSV/MERS by western blotting. And VSV/MERS specifically transduced cells expressing human dipeptidyl peptidase 4, a receptor for MERS-CoV. To investigate the immunogenicity of VSV/MERS, mice were immunized intramuscularly with VSV/MERS and alum adjuvant. MERS-CoV S-specific IgG was detected in serum samples from immunized mice. These antibodies inhibited MERS entry in vitro, suggesting a protective efficacy of VSV/MERS immunity. The data demonstrate that VSV/MERS has potent immunogenicity and could serve as a novel potential vaccine platform for MERS in dromedary camels and human.

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.

BMP-2 is a well-known TGF-beta related growth factor, having a significant role in bone and cartilage formation. It has been employed to promote bone formation in some clinical trials, and to differentiate mesenchymal stem cells into osteoblasts. However, it is difficult to obtain this protein in its soluble and active form. hBMP-2 is expressed as an inclusion body in the bacterial system. To continuously supply hBMP-2 for research, we optimized the refolding of recombinant hBMP-2 expressed in E. coli, and established an efficient method by using detergent and alkali. Using a heparin column, the recombinant hBMP-2 was purified with the correct refolding. Although combinatorial refolding remarkably enhanced the solubility of the inclusion body, a higher yield of active dimer form of hBMP-2 was obtained from one-step refolding with detergent. The refolded recombinant hBMP-2 induced alkaline phosphatase activity in mouse myoblasts, at ED50 of 300-480ng/ml. Furthermore, the expressions of osteogenic markers were upregulated in hPDLSCs and hDPSCs. Therefore, using the process described in this study, the refolded hBMP-2 might be cost-effectively useful for various differentiation experiments in a laboratory.

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.

시스타틴(cystatin: CST)은 C1A류 시스테인 단백질분해효소에 대한 경쟁적 가역억제자로서 동식물류에서 파파인과 같은 캐셉신을 억제 대상으로 작용하게 된다. 바이러스 유래 CST (CpBV-CST1)이 폴리드나바이러스의 일종인 CpBV (Cotesia plutellae bracovirus)에서 동정되었 다. 기존 연구는 이 유전자의 과발현이 배추좀나방(Plutella xylostella) 유충의 면역 및 발육을 교란한다는 것을 보여 주었다. 본 연구는 이 유전자 의 단백질 기능을 분석하기 위해 세균발현시스템을 이용하여 재조합단백질(rCpBV-CST1)을 형성하여 단백질분해효소에 대한 활성억제효과를 결정하고, 곤충의 면역과 발육에 대한 생리적 억제효과를 분석했다. 이 유전자 번역부위는 138 개 아미노산으로 약 15 kDa 크기의 단백질로 추 정되었다. CpBV-CST1이 먼저 pGEX 발현벡터에 재조합되고, BL21 STAR (DE3) competent cells에 형질전환된 후 0.5 mM IPTG로 4 시 간동안 과발현되었다. 분리된 재조합단백질은 파파인에 대한 뚜렷한 억제효과를 나타냈다. 이 재조합단백질은 파밤나방(Spodoptera exigua)에 대 해서 혈구소낭형성의 세포성 면역반응을 억제하고, 경구로 처리할 때 배추좀나방의 유충발육을 처리 농도에 비례하여 제한시켰다. 이상의 결과 는 CpBV-CST1이 해충 밀도 억제에 응용될 수 있음을 제시하고 있다.

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.

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.

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.

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.

Abnormal prions are infectious agents involved in a neuro-degenerative disease, which occurs naturally such as Chronic Wasting Disease (CWD) in deer and elk, Bovine Spongiform Encephalopathy (BSE) in cattle, Scrapie in sheep and goats and Creutzfeldt-Jakob Disease (CJD) in humans. The cellular prion protein of the elk consists of 233 amino acids (residues 25-257), which represents an autonomous folding unit with three α-helices and two-stranded anti-parallel β-sheets. Here, we demonstrated elk-recPrP (Elk recombinant prion protein) which can be obtained as follows; (1) Cloning of elk PrP gene, (2) Expression of a histidine-tagged full-length elk PrP by induction with IPTG in E. coli and (3) Purification by affinity chromatography using Ni-NTA agarose resin. In Western blot and ELISA analysis, elk-recPrP showed specific activity against anti-PrP monoclonal antibody. Thus, our elk-recPrP would be a useful tool for the understanding of basic structure and mechanism studies of PrPSC formation.

Human recombinant IL-32 induces the production of large amounts of several proinflammatory cytokines and chemokines, even in macrophage cell lines by activation of NF-κB and MAPK. The ability of IL-32 to potentiate inflammation is not the sole action of this cytokine. IL- 32 stimulates prostaglandin (PG)E2 in human PBMCs, which are pivotal in inflammation. The development of recombinant protein consisting of IL-32 is of great medical and industrial importance. Agrobacterium strains GV3101 harboring pCAMBIA1304 include IL- 32 vectors were used to infect the mushroom fruiting body pieces. For transformation experiments, fruiting body gill tissue pieces were vacuum infiltrated with the suspension of induced bacteria until the air had been completely purged. Mushroom mycelium appeared at the margins of the tissue pieces after 9 to 14 days on selection medium with hygromycin at 50㎍/㎖. Transformants PCR analyses confirmed that the IL-32 gene was into the genome of Pleurotus eryngii. The present results demonstrating the usefulness of the transgenic technique s in the genetic manipulation and improvement of mushroom.