• Asian-Australasian Journal of Animal Sciences
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• v.29 no.1
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• pp.126-133
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• 2016

A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-${\beta}$-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) $DH5{\alpha}$. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli $DH5{\alpha}$ harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine.Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was $55^{\circ}C$, but it retained over 90% of maximum activity in a broad temperature range ($40^{\circ}C$ to $60^{\circ}C$). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, $K_m$ and $V_{max}$ of rEG1 were 0.39% CMC and 143 U/mg, respectively.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.205-209
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• 2008

This study was carried out to develop knock-in vector for EGFP (enhanced green fluorescent protein) expression in porcine $\beta$-casein locus. For construction of knock-in vector using porcine $\beta$-casein gene, we cloned the $\beta$-casein genome DNA from porcine fetal fibroblast cells, EGFP and SV40 polyA signal using PCR. The knock-in vectors consisted of a 5-kb fragment as the 5' recombination arm and a 2.7-kb fragment as the 3' recombination arm. We used the neomycin resistance gene ($neo^{r}$) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. To demonstrate EGFP expression from knock-in vector, we are transfected knock-in vector that has EGFP gene in murine mammary epithelial cell line HC11 cells with pSV2 neo plasmid. The EGFP expression was detected in HC11 cells transfected knock-in vector. This result demonstrates that this knock-in vector may be used for the development of knock-in transgenic pig.

• Parasites, Hosts and Diseases
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• v.52 no.1
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• pp.93-97
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• 2014

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl ${\beta}$-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.

• Korean Journal of Veterinary Research
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• v.46 no.4
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• pp.337-345
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• 2006

The shigellae are common etiological agents of bacillary dysentery in humans and primates. During four years from 2002 to 2005, 22 strains of Shigella spp. were isolated from the diarrheic patients in Seoul region. All of them were identified as S. flexneri by biochemical tests and serotyping. The prevalence of serotypes were variable by year, but the major serotypes were 2a and 3a. In an antimicrobial susceptibility test, all of the isolates were resistant to streptomycin and tetracycline, and susceptible to amikacin, kanamycin, cefoxitin, and gentamicin. All of the isolates showed the multi-resistant patterns over 3 drugs. By analysis of the plasmid profile the isolates were classified into 7 groups (P1~P7). Serotypes 2a and 2b were distributed to P1, P2, P3, and P4. Serotype 3a was differentiated to P5 and serotype 3b, to P6 and serotype 4a, to P7. PCR results showed that all isolates were positive for two virulence genes, ipaH and ial, but none of the strains had stx gene. The set1A and set1B genes were detected from 12 isolates (54.5%) that belonged to serotype 2a and 2b. The sen gene was detected from 19 isolates (86.4%). The 22 isolates showed 12 to 17 DNA fragments in the sizes ranging from 20.5 kb to 1135 kb, resulting in 13 patterns by the PFGE with Not I digestion. The PFGE patterns of the isolates showed the close relation with the serotypes, but no relations with year of isolation and antimicrobial resistance.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.267-273
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• 1993

We screened promoters inducible by superoxide radical from Escherichia coli. For this. we constructed random promoter library from E. coli MG 1655 using a promoter-probing plasmid. pJAC4. Six hundred and sixty clones in this library were classified based on their promoter strength by ampicillin gradient plate assay. Three hundred and eighty three clones with relatively weak to medium promoter strength were selected and then screened for their inducibility by superoxide radical on ampicillin gradient plate containing paraquat. Three clones (clones 5. 15 and 34) were detected to be induced by paraquat treatment and the level of induction were between 1.4 and 4 folds. Comparison of nucleotide sequences of the cloned promoter fragment with registered sequences in GENBANK and EMBL databases suggests that the cloned DNA fragments have not been yet characterized in E. coli. Transcription start sites in these clones were determined by rrimer extension and S I nuclease protection analysis. S 1 analysis of clones 5 and IS indicated that the mRNA levels were increased by paraquat treatment. Especially. clone 5 \vas found to have two transcription start sites. the upstream start site of which was selectively used by paraquat treatment. Searching for promoter clements. we found that only the downstream promoter of clone 5 has -10 and - 35 promoter elements recognized by RNA polymerase ($E\sigma^{70}$) and the others have no conserved promoter elements. This suggests that these superoxideinducible promoters may require transcription initiation protein(s) other than $E\sigma^{70}$.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.145-152
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• 2007

Transgenic mice were generated by microinjecting a plasmid DNA containing the SV40 (simian virus 40) large T antigen (Tag) gene fused with mouse albumin promoter/enhancer sequences into fertilized one-cell mouse embryos. Among eleven founder transgenic animals, four developed hepatocellular carcinoma, two showed kidney cancer and one developed skin and brain tumors. Three stable transgenic lines, #1-2, #1-6 and #1-11 were established. Members of the lines #1-6 and #1-11 reproducibly developed liver tumors by 8 to 10 weeks of age but did not exhibit any phenotypic changes in other tissues. Histological changes loading to liver tumor formation occurred with predictable kinetics and could be classified into three distinct stages; (a) newborn to 3 weeks of age, characterized by hyperplastic hepatocytes with reduced amounts of cytoplasm without any nuclear alterations, (b) between 4 to 8 weeks of age, characterized by diffuse liver cell dysplasia without observable tumor nodules, and (c) 9 weeks of age and thereafter, characterized by hepatocellular carcinomas in the background of extensive liver dysplasia. Metastasis to the lung from a liver carcinoma was observed in #1-11 founder animal. This transgenic mouse system displays similarities with human liver cancers in a number of aspects and provides a useful model for the study of molecular events involved in hepatocarcinogenesis.

• Applied Biological Chemistry
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• v.38 no.2
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• pp.111-117
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• 1995

In Escherichia coli, CRP forms a complex with cAMP and acts as a transcriptional regulator of many genes, including sugar metabolism operons. The E. coli MK2001, which is introduced the altered crp, is functional in the expression of lac, ara and man, in the absence of cAMP. However, the expression of mal gene is fully activated by the addition of cAMP or cGMP. The object of the study is cloning of the sfs (sugar fermentation stimulation) genes, which was involved in regulation of mal gene expression with the altered crp gene, and structural analysis and characterization of the genes at the molecular level. We have cloned 5 different E. coli genes which stimulate the maltose metabolism in a crp, cya::km (MK2001) background. Newly identified genes were designated as sfs. One of the sfs genes (pPC1), located at the 53.2 min map position on the E. coli chromosome, was further analyzed. Expression of the genes, which is involved in maltose metabolism, malQ (amylomaltase), was increased to 5.8-fold in the presence of a plasmid, pAP5, containing the subcloned sfs4 gene. The nucleotide seguence of a common 2,126 bp segment of the pPCM1 was determined and two open reading frames (ORF1 and ORF2) were detected. The ORF1 encodes the sfs4 gene and ORF2 encodes a truncated protein. Potential CRP binding site is located in the upstream of the putative promoter in the regulatory region. Expression of the cloned sfs4 gene was positively regulated by the cAMP-CRP complex.

• The Korean Journal of Food And Nutrition
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• v.17 no.1
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• pp.47-52
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• 2004

In this study, I attempted to develope the expression and purification system of human mammaglobin proteins in Escherichia coli and to produce anti-human mammaglobin rabbit antibody for the detection of human mammaglobin protein in the peripheral blood of breast cancer patients. Human mammaglobin gene was cloned and sequenced from m-RNAs purified from donated breast cancer tissues using RT-PCR. The cloned gene was inserted into pET30, pET22, and pET32 plasmid. The cloned gene in pET30 yields insoluble proteins which was difficult to purify from the cells extracts. The mammaglobin gene in pET32 was strongly expressed soluble proteins which were isolated using Ni-NTA affinity chromagraphy and DEAE-ion exchange chromatography, followed by enterokinase digestion of the purified proteins. The isolated proteins had enough purity to use as a antigen for the production of anti-mammaglobin antibody in rabbits. The polyclonal antibody produced against the isolated mammaglobin showed a specificity to mammaglobin after Westernblot immuno assay. In conclusion, the isolated mammaglobin protein and the anti-mammaglobin rabbit antibody may be used for diagnosis of breast cancer as well as development of anti-breast cancer drug.

• Journal of Life Science
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• v.25 no.2
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• pp.223-230
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• 2015

Regulator of calcineurin 1 (RCAN1) is an endogenous calcineurin inhibitor that plays an important role in the pathogenesis of diseases related to the calcineurin-NFATc1 signaling pathway. The RCAN1-4 isoform is subject to NFATc1-dependent regulation. During receptor activator of nuclear factor kappa-B ligand (RANKL)-stimulated osteoclastogenesis, the calcineurin-NFATc1 pathway is critical. Because there is little information available on the role of RCAN1 in osteoclast differentiation, this study investigated whether changes in RCAN1 expression are related to the calcineurin-NFATc1 pathway and osteoclast differentiation. Mouse bone marrow monocytes (BMMs) were treated with 50 ng/ml of RANKL and M-CSF. Expression levels of NFATc1, calcineurin, and RCAN1 isoforms were determined using RT-PCR and Western blotting. Osteoclast differentiation was examined using tartrate-resistent acid phosphatase (TRAP) staining. To evaluate the effect of RCAN1 overexpression on osteoclastogenesis, cells were transfected with a mouse RCAN1-4 cDNA plasmid. After RANKL stimulation of BMMs, expression of NFATc1 and RCAN1 was increased at the mRNA and protein level, while calcineurin expression was unchanged. When the RCAN1-4 gene construct was transfected, the expression of RCAN1 protein was not increased despite several-fold increases in RCAN1-4 mRNA expression. Regardless of RANKL stimulation, over-expression of RCAN1-4 tended to reduce NFATc1 expression and knock-down of RCAN1 increase it. While BMMs transfected with the RCAN1-4 vector were differentiated into distinct osteoclasts, their phenotypes did not vary from those of mock controls. These results suggest that RCAN1 has a limited effect on the calcineurin-NFATc1 pathway during RANKL-stimulated osteoclast differentiation.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.