• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.676-684
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• 1998

Baculovirus transfer and expression vectors with Hyphantria cunea nuclear polyhedrosis virus (HcNPV) were constructed. An initial transfer vector, pHcEV, constructed using HcNPV was previously reported (Park et al. 1993. J. Kor. Soc. Viral. 23: 141-151). Herein, the size of the vector was properly reduced, and a functionally perfect vector was constructed and named pHcEV-IV (6.7 kb). The vector has a 2.2-kb HcNPV DNA sequence in the 5'-flanking region of the vector's polyhedrin gene promoter. The 1.8-kb HcNPV DNA sequence, poly A signal sequence, T3 primer sequence, and 13 multicloning site sequences, in order, were ligated in front of the translation start codon of the polyhedrin gene. The cloning indicating marker lacZ gene was inserted into the pHcEV-IV, named pHcEV-IV-lacZ, and transferred into the wild-type virus. Recombinant expression virus, lacZ-HcNPV, was constructed by replacing the lacZ gene in the pHcEV-IV-lacZ with the polyhedrin gene of the wild-type virus. The recombinant virus was isolated from blue plaques that produce $\beta$-galactosidase without polyhedra. The lacZ gene insertion was confirmed by Southern hybridization analysis. The expression of the lacZ gene in Spodoptera frugiperda cells infected with the lacZ-HcNPV was examined by SDS-PAGE and colorimetric assay. One 116-kDa LacZ protein band appeared on the PAGE. The production rate of the $\beta$-galactosidase was approximately 50 international units (IU) per min per ml between 2 to 5 days postinfection (p.i.). The highest activity occurred at five days p.i. was 170 IU/min/$m\ell$. The enzyme activity first appeared about 20 h p.i. as measured by colorimetric assay.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.547-552
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• 2004

Streptomyces thermovulgans malate synthase (stMS) is known to be more thermostable and thermoactive than S. coelicolor malate synthase (scMS). Therefore, based on the amino acid sequence of stMS, 3 scMS mutants, namely P186R, T8PL9P, and T8PL9PP186R, were created by site-directed mutagenesis in an attempt to engineer a more thermoactive and thermostable enzyme. An enzymatic analysis of the wild-type and mutant MS revealed that P186R and T8PL9PP186R were more thermoactive than the wild-type scMS and T8PL9P. Furthermore, all 3 mutants exhibited a greater thermo stability than scMS, thereby suggesting that both R186 and P8P9 can cause increased thermo stability in scMS.

• Journal of Applied Biological Chemistry
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• v.43 no.1
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• pp.12-17
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• 2000

To understand the signal transduction pathway that leads to the activation of the wound-inducible proteinase inhibitor II (pin2) promoter. $F_2$ progenies of wound (-) mutant crossed with wild-type Arabidopsis plants were biochemically and genetically characterized. Wound (-) mutant was derived from transgenic Arabidopsis plants containing bacterial cytosine deaminase gene under the control of pin2 promoter. The cytosine deaminase assays indicated that wound (-) mutant is a dominant inhibitor of wound-inducibility as only 3 of the $20F_2$ progenies showed cytosine deaminase (CDase) activity, To construct a structural map of the wound (-) mutant chromosomal regions, cleaved, amplified polymorphic sequences (CAPS) markers that cover all Chromosomes were used. Chromosomal regions covered by three different CAPS markers could be candidates for further fine mapping of the location of the wound (-) mutation. g4026, RGA1 and ASA1 located at 84.9 on recombinant inbred (RI) map of chromosome I, at 1.75 on RI map of chromosome II, and 18.35 on RI map of chromosome V, respectively.

• International Journal of Industrial Entomology and Biomaterials
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• v.3 no.1
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• pp.75-81
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• 2001

We have constructed a novel recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV) producing the green fluorescent polyhedra. For the production of the fluorescent polyhedra, partial polyhedrin gene containing KRKK as nuclear localization site from the BmNPV polyhedrin gene and the green fluorescent protein (gfp) gene were introduced under the control of p10 promoter of BmNPV. The recombinant BmNPV was stably produced fluorescent polyhedra in the infected Bm5 cells and the morphology of the fluorescent polyhedra was similar to that of wild-type BmNPV. The fluorescent polyhedra had 32 kDa native polyhedrin and 41 kDa fusion protein. From these data, we have further developed a novel BmNPV p10-based transfer vector producing recombinant polyhedra with foreign gene Product. The novel BmNPV P10-based transfer vector is composed of partial polyhedrin gene, factor Xa, and multiple cloning sites.

• BMB Reports
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• v.31 no.3
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• pp.287-295
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• 1998

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-Ieucine, L-isoleucine, and L-valine. The sulfonylurea-resistant ALS gene from Nicotiana tabacum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS3 was used to transform Escherichia coli strain XL1-Blue, and the mutant tobacco ALS (mALS) was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). The fusion product GST-mALS was purified in a single step on a glutathione-Sepharose column. ALS activities of 0.9-2.5 ${\mu}mol/min/mg$ protein were observed in the GST-mALS, and the Km values for pyruvate, FAD, and TPP were 10.8-24.1, $(1.9-8.9){\times}10^{-3}$, and 0.14-0.38 mM, respectively. The purified GST-mALS was resistant to both the sulfonylurea and the triazolopyrimidine herbicides, and lost its sensitivity to end products, L-valine and L-leucine. For comparision, the tobacco wild-type recombinant ALS fused with GST, GST-wALS, was also characterized with respect to its pyruvate and cofactor bindings. These results suggest that the purified mutant recombinant tobacco ALS was functionally active, that the mutations resulting in herbicide resistance has affected pyruvate and cofactor bindings," and that the two classes of herbicides interact at a common site on the plant ALS.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.27-34
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• 2006

This study was conducted to compare the reproduction ability of the wild type boar and recombinant human erythropoietin (hEPO) transgenic boar semen. Ejaculated boar semen was analyzed by flow cytometry, Elisa and IVF methods. In experiment 1, flow cytometric analysis showed that the live sperm ratio of transgenic boar sperm significantly lower (P<0.05) than that of wild type boar after incubation at 20, 22, 24 and 26 hr. In experiment 2, the presence and levels of various cytokines (IL-6, IL-10 and $TNF-{\alpha}$) to related animal reproduction in the seminal and blood plasma were examined using specific enzyme immunoassay. There was no significant difference between both groups. In experiment 3, the fertilizing capacity and developmental ability of both boar sperm were compared. The transgenic boar sperm had a significantly low capacity of penetration, sperm-zona binding, embryo development, and blastocyst formation compared to wild type sperm (P<0.05). These results suggest that transgenic boar sperm harboring hEPO gene has low sperm viability than wild type boar, and it is a reason to decrease of fertility and litter size.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.29-37
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• 1997

To use recombinant viruses with wider host range as viral insecticides, we investigated the characteristics and pathogenicity of host range expanded recombinant viruses in insect cells. We compared host range expanded recombinant viruses, RecS-B6 and RecB-8, constructed by cotransfection of Autographa californica nuclear polyhedrosis virus (AcNPV) and Bombyx mori NPV (BmNPV), to host range expanded AcNPV, Ac-BH, by substitution of the 0.6 Kb fragment of the BmNPV helicase gene. Restriction endonuclease profiles of RecS-B6 and RecB-8 DNAs were different from those of parent viruses. Nucleotide sequence analysis of the 0.6 Kb region in the putative helicase gene of RecS-B6 and RecB-8 showed that their structures were identical to the counterpart region of BmNPV. Comparison of viral replication of these recombinant viruses in Sf-21 and BmN-4 cells showed that Ac-BH, compared to wild type viruses, replicated well in BmN-4 cells but poorly in Sf-21 cells. In contrast, RecS-B6 and RecB-8 replicated relatively well in both cells compared to parent viruses. These results may imply that random genomic recombinant viruses, RecS-B6 and RecB-8, possess better potential as viral pesticides than helicase-mediated recombinant virus, Ac-BH.

• Journal of Microbiology and Biotechnology
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• v.26 no.7
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• pp.1173-1181
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• 2016

Salmonella spp. are gram-negative flagellated bacteria that cause a variety of diseases in humans and animals, ranging from mild gastroenteritis to severe systemic infection. To explore development of a potent vaccine against Salmonella infections, the gene encoding outer membrane protein L (ompL) was inserted into the swinepox virus (SPV) genome by homologous recombination. PCR, western blot, and immunofluorescence assays were used to verify the recombinant swinepox virus rSPV-OmpL. The immune responses and protection efficacy of rSPV-OmpL were assessed in a mouse model. Forty mice were assigned to four groups, which were immunized with rSPV-OmpL, inactive Salmonella (positive control), wild-type SPV (wtSPV; negative control), or PBS (challenge control), respectively. The OmpL-specific antibody in the rSPV-OmpL-immunized group increased dramatically and continuously over time post-vaccination, and was present at a significantly higher level than in the positive control group (p < 0.05). The concentrations of IFN-γ and IL-4, which represent Th1-type and Th2-type cytokine responses, were significantly higher (p < 0.05) in the rSPV-OmpL-vaccinated group than in the other three groups. After intraperitoneal challenge with a lethal dose of Salmonella typhimurium CVCC542, eight out of ten mice in the rSPV-OmpL-vaccinated group were protected, whereas all the mice in the negative control and challenge control groups died within 3 days. Passive immune protection assays showed that hyperimmune sera against OmpL could provide mice with effective protection against challenge from S. typhimurium. The recombinant swinepox virus rSPV-OmpL might serve as a promising vaccine against Salmonella infection.

• Journal of fish pathology
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• v.37 no.1
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• pp.17-23
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• 2024

Spring viremia of carp virus (SVCV) poses a significant threat to numerous cyprinid fish species, particularly the common carp (Cyprinus carpio), often resulting in substantial mortalities. This study explores the potential use of a chimeric recombinant snakehead rhabdovirus carrying the SVCV G gene (rSHRV-Gsvcv) as a live vaccine against SVCV infection. Through virulence testing in zebrafish at different temperatures (15 ℃ and 20 ℃), no mortality was observed in groups infected with either rSHRV-wild or chimeric rSHRV-Gsvcv at both temperatures, whereas 100% mortality occurred in fish infected with wild-type SVCV. Subsequently, as no mortality was observed by rSHRV-Gsvcv, three independent experiments were conducted to determine the possible usage of chimeric rSHRV-Gsvcv as a vaccine candidate against SVCV infection. Fish were immunized with either rSHRV-Gsvcv or rSHRV-wild, and their survival rates against the SVCV challenge were compared with a control group injected with buffer alone at four weeks post-immunization. The results showed that chimeric rSHRV-Gsvcv induced significantly higher fish survival rates compared to rSHRV-wild and the control groups. These findings suggest that genetically engineered chimeric rSHRV-Gsvcv holds the potential for a prophylactic measure to protect fish against SVCV infection.

• The Journal of Korean Society of Virology
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• v.28 no.1
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• pp.71-78
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• 1998

Vaccinia virus is the prototype orthopoxvirus that has been used as a vaccine strain for small pox. This virus has been used to express a variety of cellular and viral genes in mammalian cells at high levels. Interleukin-4 (IL-4) has been found to stimulate the proliferation of T cells and enhance the cytolytic activity of cytotoxic T lymphocytes. To test the immunotherapeutic potential of IL-4 delivered in vivo by poxvirus, a recombinant vaccinia virus expressing the murine IL-4 gene (RVVmIL-4) was constructed. A high level of IL-4 production was confirmed by infecting HeLa cells and measuring IL-4 in cell culture supernatant by ELISA. As a tumor model, two cell lines were used; the murine T leukemic line P388 and the murine breast cancer line TS/A. CDF1 mice were intraperitoneally inoculated with $1\;{\times}\;10^5$ cells of P388. Mice were injected at the same site with $5\;{\times}\;10^5\;PFU$ of recombinant vaccinia virus; first, 3 days after the injection of tumor cells and thereafter once every week for 3 weeks. Intraperitoneal injections of RVVmIL-4 significantly prolonged the survival time of mice inoculated with tumor cells. All mice injected with RVVmIL-4 remained alive for 30 days after the postinoculation of tumor cells, while 100% and 70% of the animals injected with saline or wild type vaccinia virus died, respectively. In another tumor model using TS/A, tumor was established by subcutaneously inoculating $2{\times}10^5$ tumor cells to BALB/c mice. After tumor formation was confirmed on day 4 in all mice, $5\;{\times}\;10^6\;PFU$ of RVVmIL-4 was inoculated subcutaneously three times, once every week for 3 weeks. The TS/A tumor was eradicated in two of the nine mice. Seven of the nine mice treated with RVVmIL-4 developed a tumor, but tumor growth was significantly delayed compared to those treated with saline or wild type vaccinia virus. These results indicate that recombinant vaccinia viruses may be used as a convenient tool for delivering immunomodulator genes to a variety of tumors.