• Journal of Life Science
• /
• v.19 no.7
• /
• pp.845-851
• /
• 2009

A vector harboring double cassettes; a heterologous gene expression cassette of pHCE-InaN-antigen and a ghost formation cassette of pAPR-cI-E lysis 37 SDM was constructed and introduced to E. coli DH5a. For the production of a bacterial ghost vaccine, bacterial ghosts from E. coli / Streptococcus iniae with four different types of antigens - enolase, GAPDH, sagA and piaA - were produced by the optimization of fermentation parameters such as a glucose concentration of 1 g/l, agitation of 300 rpm and aeration of 1 vvm. Efficiency of ghost bacteria formation was evaluated with cultures of OD$_{600}$=1.0, 2.0 and 3.0. The efficiency of the ghost bacteria formation was 99.54, 99.67, 99.99 and 99.99% with inductions at OD$_{600}$=3.0, 1.0, 2.0 and 1.0 for E. coli/S. iniae antigens enolase, piaA, GAPDH and sagA, respectively. Ghost bacteria as a vaccine was harvested by centrifugation. The antigen protein expressions were analyzed by SDS-PAGE and western blot analysis, and the molecular weights of the enolase, piaA, GAPDH and sagA were 78, 26, 67 and 26 kDa, respectively. The molecular weights of the expressed antigens were consistent with theoretical sizes obtained from the amino acid sequences.

• Korean Journal of Microbiology
• /
• v.32 no.4
• /
• pp.271-279
• /
• 1994

To develop a yeast strain which stably secretes both $\alpha$-amylase and glucoamylase and therefore is able to convert starch directly to ethanol, a mouse salivary $\alpha$-amylase cDNA gene with a yeast alcohol dehydrogenase I promoter has been introduced into the cell of a Saccharomyces diactaticus hybrid strain secreting only glucoamylase. To secrete both enzymes more stably without loss of the $\alpha$-amylase gene during a cell-multiplication, an integrating plasmid vector containing $\alpha$-amylase gene was constructed and introduced into the yeast cell. The results showed that the linearized form of the integrating vector was superior in the transformation efficiency and the rate of the expression of the $\alpha$-amylase gene than the circular type of the vector. The yeast transformant having a linearized plasmid vector exhibited higher mitotic stability than the yeast transformant habouring episomat plasmid vector. The transformant containing the linearized vector producing both $\alpha$-amylase and glucoamylase exhibited 2-3 times more amylolytic activity than the original untransformed strain secreting only glucoamylase.

• Journal of Embryo Transfer
• /
• v.32 no.3
• /
• pp.73-79
• /
• 2017

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous ${\alpha}1,3$-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig ($GalT^{-MCP/+}$), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig ($GalT^{-MCP/-MCP}$) by crossbreeding $GalT^{-MCP/+}$ pigs. Two female founders gave birth to six of $GalT^{-MCP/-MCP}$, and seven $GalT^{-MCP/+}$ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the $GalT^{-MCP/-MCP}$ pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the $GalT^{-MCP/-MCP}$ pig is a useful candidate to apply xenotransplantation study.

• Microbiology and Biotechnology Letters
• /
• v.42 no.2
• /
• pp.190-193
• /
• 2014

CMP-Neu5Ac synthetase is a key enzyme for the synthesis of CMP-Neu5Ac, which is an essential precursor of sialylated glycoconjugates. For the soluble expression of the CMP-Neu5Ac synthetase gene (neuA) from Escherichia coli K1, various heat shock proteins were co-expressed in E. coli BL21 (DE3) Star. In order to do this, a pG-KJE8 plasmid, encoding genes for GroEL-ES and DnaK-DnaJ-GrpE, was co-transformed with neuA and was expressed at $20^{\circ}C$ by the addition of 0.01 mM IPTG and 0.005 mg/ml L-arabinose. The co-expression of a variety of heat shock proteins resulted in the remarkably improved production of soluble CMP-Neu5Ac synthetase in E. coli.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.7
• /
• pp.1326-1334
• /
• 2008

The prime-boost vaccination with DNA vaccine and recombinant viral vector has emerged as an effective prophylactic strategy to control infectious diseases. Here, we compared the protective immunities induced by multiple alternating immunizations with DNA vaccine (pCIgB) and replication-incompetent adenovirus (Ad-gB) expressing glycoprotein gB of pseudorabies virus (PrV). The platform of pCIgB-prime and Ad-gB-boost induced the most effective immune responses and provided protection against virulent PrV infection. However, priming with pCIgB prior to vaccinating animals by the DNA vaccine-prime and Ad-boost protocol provided neither effective immune responses nor protection against PrV. Similarly, boosting with Ad-gB following immunization with DNA vaccine-prime and Ad-boost showed no significant responses. Moreover, whereas the administration of Ad-gB for primary immunization induced Th2-type-biased immunity, priming with pCIgB induced Th1-type-biased immunity, as judged by the production of PrV-specific IgG isotypes and cytokine IFN-$\gamma$. These results indicate that the order and injection frequency of vaccine vehicles used for heterologous prime-boost vaccination affect the magnitude and nature of the immunity. Therefore, our demonstration implies that the prime-boost protocol should be carefully considered and selected to induce the desired immune responses.

• Journal of Dairy Science and Biotechnology
• /
• v.21 no.2
• /
• pp.97-104
• /
• 2003

It is extremely important to destroy the antigenicity of milk proteins for dietetic treatment of infants with milk allergy. Enzymatic digestion of milk protein is not only effective for destroying antigenicity, but it also is less liable to alter the nutritive value. Bovine casein was hydrolyzed with eight different commercial proteases derived from bacterias or fungi, either individually or in combination to eliminate protein allergenicity. The average molecular weight of casein hyrdolysates determined by size exclusion chromatography is about 550${\sim}$2,300 dalton range. Antigenicity of the casein hyrdolysates was not detected by heterologous passive cutaneous anaphylaxis in guinea pig-rabbit antiserum system. The inhibition test on the enzyme-linked immunosorbent assay(ELISA) showed that the antigenicity of casein hydrolysates is lowed up to 1/8,000 than that of intact bovine casein. As the enzyme reaction was carried out by the combination of bacterial and fungal protease, casein hydrolysates showed much lower bitterness and antigenicity. It suggests that these hydrolysates will be applied to many kinds of foods including the development of hypo-allergenic infant formula.

• Journal of Chest Surgery
• /
• v.29 no.11
• /
• pp.1182-1190
• /
• 1996

The best treatment of congenital or acquired tracheal stenosis is resection and end to end anastomosis. Various prosthetic material and tissue graft replacement can be considered when the stenotic segment is too long, but their uses are still limited due to many serious complications. The present study examined the effect of immunosuppression and cryopreserved allograft trachea after intraperitoneal omental implantation for evaluation of the possibility of tracheal transplantation. Thirty tracheal segments were harvested from fifteen donor Wistar rats. Among them eighteen segments were implanted immediately(group I, II, III) and twelve segments were used for cryopreservation(group IV, V). Heterotopical intraperitoneal implantation was performed in five groups of rats(n=6); Group I was Wistar syngeneic controls and received no immunosuppression. Group II and III were those of Sprague-Dawley recipients, the former receiving no immunosuppression and the latter receiving immunosuppression(Cyclosporin A 15mg/kg/day, Methylprednisolone 2mg/kg/day). Group IV and V were groups of Sprague-Dawley recipients, the former receiving immunosuppression and the latter receiving no Immunosuppression. After 28 days, rats were sacrificed and the tracheal segments were histologically evaluated. Epithelial thickness was significantly decreased in group II, IV. Epithelial regeneration score was also significantly decreased in II. All rats maintained well their round tracheal contour. In conclusion; I) trachea could be preserved for a long time with cryo method, 2) epithelium could regenerate fully with omentopexy in cryopreserved trachea, 3) immunosuppresion was not necessary with cryopreserved trachea.

• Molecules and Cells
• /
• v.27 no.4
• /
• pp.475-480
• /
• 2009

The transcription of soybean (Glycine max) calmodulin isoform-4 (GmCaM-4) is dramatically induced within 0.5 h of exposure to pathogen or NaCl. Core cis-acting elements that regulate the expression of the GmCaM-4 gene in response to pathogen and salt stress were previously identified, between -1,207 and -1,128 bp, and between -858 and -728 bp, in the GmCaM-4 promoter. Here, we characterized the properties of the DNA-binding complexes that form at the two core cis-acting elements of the GmCaM-4 promoter in pathogen-treated nuclear extracts. We generated GUS reporter constructs harboring various deletions of approximately 1.3-kb GmCaM-4 promoter, and analyzed GUS expression in tobacco plants transformed with these constructs. The GUS expression analysis suggested that the two previously identified core regions are involved in inducing GmCaM-4 expression in the heterologous system. Finally, a transient expression assay of Arabidopsis protoplasts showed that the GmCaM-4 promoter produced greater levels of GUS activity than did the CaMV35S promoter after pathogen or NaCl treatments, suggesting that the GmCaM-4 promoter may be useful in the production of conditional gene expression systems.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
• /
• 2019.05a
• /
• pp.533-536
• /
• 2019

Recombinant baculoviruses are widely used to express heterologous genes in cultured insect cells. Recombinant baculoviruses can serve as gene-transfer vectors for expression of recombinant proteins in a wide range of mammalian cell types. Baculovirus system has significant benefits in view of safety, large-scale, and high level of gene expression. In this study, baculoviral vectors which were reconstructed from pOPINEneo-3C-GFP vector, were recombined with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP), and p53 with NcoI and XhoI. These recombinant vectors were infected with various cells and cell lines. The baculovirus vector thus developed was analyzed by comparing the metastasis and expression of the recombinant genes with conventional vectors. These results suggest that the baculovirus vector has higher efficiency in metastasis and expression than the control vector. This work was supported by a grant from Mid-Career Researcher Program(NRF-2016R1A2B4016552) through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(MSIP).

• Journal of Life Science
• /
• v.31 no.1
• /
• pp.83-89
• /
• 2021

Uu-ilys, an i-type lysozyme from spoon worm (Urechis unicinctus), is an innate immune factor that plays an important role in the defense against pathogens. It also possesses non-enzymatic antibacterial activity. Thus, there is a possibility to develop an antimicrobial model peptide from Uu-ilys. In this study, we report the design, production, and antibacterial activity of an Uu-ilys analog that exhibits antibacterial activity. The Uu-ilys structure was fragmented according to its secondary structures to predict the regions with antimicrobial activity using antimicrobial peptide (AMP) prediction tools from different AMP databases. A peptide containing the C-terminal fragment was predicted to exert antimicrobial activity. The chosen fragment was designated as an Uu-ilys analog containing the C-terminal fragment, Uu-ilys-CF. To examine the possibility of developing an AMP using the sequence of Uu-ilys-CF, recombinant fusion protein (TrxA-Uu-ilys-CF) was produced in an expression system that was heterologous. The produced fusion protein was cleaved after methionine leaving Uu-ilys-CF free from the fusion protein. This was then isolated through high performance liquid chromatography and reverse phase column, CapCell-Pak C18. The antibacterial activity of Uu-ilys-CF against different microbial strains (four gram-positive, six gram-negative, and one fungal strain) were assessed through the ultrasensitive radial diffusion assay (URDA). Among the bacterial strains tested, Salmonella enterica was the most susceptible. While the fungal strain tested was not susceptible to Uu-ilys-CF, broad spectrum antibacterial activity was observed.