• Journal of Life Science
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• v.18 no.11
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• pp.1592-1599
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• 2008

The $\beta$-lactamase gene was cloned into E. coli DH5$\alpha$ from Bacillus sp. J105 with strong resistance against $\beta$-lactam antibiotics. The chromosomal DNA was partially digested with Sau3AI and ligated to BamHI digested pLAFR3. $\beta$-Lactamase positive clones were obtained by using in vitro packaging kit. The pKL11-${\Delta}4.6$ with $\beta$-lactamase activity was obtained by subcloning of the recombinant plasmid ($\beta$-lac +). The 6.5 kb fragment in the subcloned plasmid was sequenced. The DNA fragment that contains the $\beta$-lactamase gene encodes 309 amino acids. The 0.17 kb upstream region was similar to those of B. thuringinesis and B. cereus with 97% identity. The deduced amino acids sequence was also similar to those of $\beta$-lactamase from B. thuringinesis and B. cereus with 97% and 94% identity, respectively. The phylogenetic tree also showed the relationships of the $\beta$-lactamase gene of Bacillus sp. J105 to genetically related that of other Bacillus strains. Analysis of expression pattern of the pKL11-${\Delta}4.6$ in E. coli, revealed that the secretion efficiency of $\beta$-lactamase was $4{\sim}5%$ and the molecular weight was as same as that of original $\beta$-lactamase (31 kDa) from Bacillus sp. J105.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.14
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• pp.6019-6026
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• 2015

Background: The aim of this study was to assess the relationship between IL-18 gene polymorphisms and HBV-related diseases and whether these polymorphisms influence its expression in the Guangxi Zhuang population. Materials and Methods: We enrolled 129 chronic HBV infected (CHB) patients, 86 HBV-related liver cirrhosis (LC) patients and 160 healthy controls in our study. Polymerase chain reaction-restriction fragment length polymorphism methods were used to detect IL-18 gene -607C/A, -137G/C polymorphisms, and an ELISA kit was employed to determine serum IL-18 levels. Results: No correlation was found between the -607C/A polymorphism and risk of HBV-related disease. For the -137G/C polymorphism, the GC genotype and C allele were associated with a significantly lower risk of CHB (95%CI: 0.32-0.95, p=0.034 and 95%CI: 0.35-0.91, p=0.018) and HBV-related LC (95%CI: 0.24-0.89, p=0.022 and 95%CI: 0.28-0.90, p=0.021). A similar decreased risk was also found with the A-607C-137 haplotype. With respect to IL-18 expression, it was significantly lower in both patient groups, but no association was noted between the two polymorphisms in the IL-18 gene and its expression. Conclusions: Our study indicated that the -137C allele in the IL-18 gene may be a protective factor for HBV-related disease, and serum IL-18 level may be inversely associated with CHB and HBV-related LC.

• Journal of the East Asian Society of Dietary Life
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• v.23 no.6
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• pp.723-732
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• 2013

We investigated the inducing effects of Rubus coreanus extract (RCE) on apoptosis and its related gene expressions in human breast cancer cells. MDA-MB-231 cells were cultured in the presence of 0, 200, 300, and $400{\mu}g/mL$ RCE for 24h. MTT assay demonstrated that relative cell viability measured a decrease in a dose-dependent manner (p<0.05). This dependency was also found in the increasing levels of cell death by a dual staining with Hoechst 33322 and propidium iodide (p<0.05). These close associations was also observed by different stages of apoptotic processes, as shown by an Apoptosis Detection Kit. To determine whether the alterations in such cell activities obtained above cause the induction of apoptotic genes, PT-PCR was performed expressions of both Bcl-2 and Bax mRNAs. The Bcl-2/Bax ratio which is an important indicator of apoptosis, was found to have significantly decreased dose dependence (p<0.05). Western blot analysis also demonstrated that Caspase-3 significantly increases in a dose-dependent manner (p<0.05) in addition to similar alterations of other proteins examined. Taken these results together, the ethanolic RCE used induces a reduction in cell viability along with increased membrane permeability. This leads to a precautious apoptotic process and, subsequently, cell death through the apoptotic pathway involving Bax and Caspase-3 in human breast cancer MDA-MB-231 cells.

• Journal of Microbiology and Biotechnology
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• v.12 no.3
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• pp.463-469
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• 2002

Rotaviruses are the world-wide leading causative agents of severe dehydrating gastroenteritis in young children and animals. The outer capsid glycoprotein VP7 and inner capsid glycoprotein VP6 of rotaviruses are highly antigenic and immunogenic. An SFV-based expression system has recently emerged as a useful tool for heterologous protein production in mammalian cells, exhibiting a much more efficient performance compared to other gene expression systems. Accordingly, the current study adopted an SFV-based expression system to express the VP7 of a group A human rotavirus from a Korean isolate, and the VP6 of a group B bovine rotavirus from a Korean isolate, in mammalian cells. The genes of the VP6 and VP7 were inserted into the SFV expression vector pSFV-1. The RNA was transcribed in vitro from pSFV-VP6 and pSFV-VP7 using SP6 polymerase. Each RNA was then electroporated into BHK-21 cells along with pSFV-helper RNA containing the structural protein gene without the packaging signal. The expression of VP6 and VP7 in the cytoplasm was then detected by immunocytochemistry. The recombinant virus was harvested by ultracentrifugation and examined under electron microscopy. After infecting BHK-21 cells with the defective viruses, the expressed proteins were separated by SDS-PAGE and analyzed by a Western blot. The results indicate that an SFV-based expression system fur the VP6 and VP7 of rotaviruses is an efficient tool for developing a diagnostic kit and/or preventive vaccine.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.16
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• pp.6581-6586
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• 2014

Background: The istone deacetylase (HDAC) inhibitor trichostatin A (TSA) is known to mediate the regulation of gene expression and antiproliferation activity in cancer cells. Kr$\ddot{u}$ppel-like factor 4 (klf4) is a zinc finger-containing transcription factor of the SP/KLF family, that is expressed in a variety of tissues and regulates cell proliferation, differentiation, tumorigenesis, and apoptosis. It may either either function as a tumor suppressor or an oncogene depending on genetic context of tumors. Aims: In this study, we tested the possibility that TSA may increase klf4 expression and cancer cell growth inhibition and apoptosis in SKOV-3 and A549 cells. Materials and Methods: The cytotoxicity of TSA was determined using the MTT assay test, while klf4 gene expression was assessed by real time PCR andto ability of TSA to induce apoptosis using a Vybrant Apoptosis Assay kit. Results: Our results showed that TSA exerted dose and time dependent cytotoxicity effect on SKOV-3 and A549 cells. Moreover TSA up-regulated klf4 expression. Flow cytometric analysis demonstrated that apoptosis was increased after TSA treatment. Conclusions: Taken together, this study showed that TSA increased klf4 expression in SKOV3 and A549 cell lines, consequently, klf4 may played a tumor-suppressor role by increasing both cell growth inhibition and apoptosis. This study sheds light on the details of molecular mechanisms of HDACI-induced cell cycle arrest and apoptosis.

• Journal of Ginseng Research
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• v.33 no.1
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• pp.55-58
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• 2009

Generally, the direct sequencing through PCR is faster, easier, cheaper, and more practical than clone sequencing. Frequently, standard PCR amplification is usually interpreted by mispriming internal or external regions of the target template. Normally, DNA fragments were eluted from the gel using Gel extraction kit and subjected to direct sequencing or cloning sequencing. Cloning sequencing has often troublesome and needs more time to analyze for many samples. Since touchdown (TD) PCR can generate sufficient and highly specific amplification, it reduces unwanted amplicon generation. Accordingly, TD PCR is a good method for direct sequencing due to amplifying wanted fragment. In plants the 5S-rRNA gene is separated by simple spacers. The 5S-rRNA gene sequence is very well-conserved between plant species while the spacer is species-specific. Therefore, the sequence has been used for phylogenetic studies and species identification. But frequent occurrences of spurious bands caused by complex genomes are encountered in the product spectrum of standard PCR amplification. In conclusion, the TD PCR method can be applied easily to amplify main 5S-rRNA and direct sequencing of panax ginseng cultivars.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6245-6248
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• 2013

Aims: To study effects of down-regulation of pituitary tumor-transforming gene (PTTG) on proliferation and metastasis ability of the SCL-1 cutaneous squamous cell carcinoma (CSCC) cell line and explore related mechanisms. Methods: SCL-1 cells were divided into 3 groups (untreated, siRNA control and PTTG siRNA). Cell proliferation assays were performed using a CCK-8 kit and proliferation and metastasis ability were analyzed using Boyden chambers. In addition, expression of MMP-2 and MMP-9 was detected by r-time qPCR and Western blotting. Results: Down-regulation of PTTG could markedly inhibit cell proliferation in SCL-1 cells, compared to untreated and control siRNA groups (P < 0.05). Real-time qPCR demonstrated that expression levels of PTTG, MMP-2 and MMP-9 in the PTTG siRNA group were 0.8%, 23.2% and 21.3% of untreated levels. Western blotting revealed that expression of PTTG, MMP-2 and MMP-9 proteins in the PTTG siRNA group was obviously down-regulated. The numbers of migrating cells ($51.38{\pm}4.71$) in the PTTG siRNA group was obviously lower than that in untreated group ($131.33{\pm}6.12$) and the control siRNA group ($127.72{\pm}5.20$) (P < 0.05), suggesting that decrease of proliferation and metastasis ability mediated by PTTG knock-down may be closely correlated with down-regulation of MMP-2 and MMP-9 expression. Conclusion: Inhibition of PTTG expression may be a new target for therapy of CSCC.

• Journal of Sericultural and Entomological Science
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• v.38 no.1
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• pp.19-24
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• 1996

To investigate the genes which is related to immune reaction of Bombyx mori, differential screening was carried out using naive and induced B. mori mRNA probe. To begin with, we constructed the cDNA library with mRNA isolated from fifth instar larvae injected with E. coli(4 X 106 cells/larva) using Uni ZAP XR vector kit. Thirty-two inducible cDNAs showing higher intensity on the induced mRNA probing membranes were selected. Partial nucleotide sequences of 29 clones were determined and their expessed sequence tags (ESTs) were produced. Nineteen ESTs in 29 ESTs were matched in GenBank database and the rest of them were found to be unknown. These unmatched ESTs were presumed to be novel genes. The nineteen ESTs contained variable genes related to biological process in Bombyx mori and four classes immune genes. Four clones, BmInc 6, 8, 18 and 27 were similar to two antibacterial peptide genes, hemolin gene and transferrin gene, respectively.

• Korean Journal of Veterinary Service
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• v.33 no.1
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• pp.13-21
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• 2010

Chicken anemia virus (CAV) has been recognized as an immunosuppressive agent and plays role as an etiological agent of multifactorial diseases in chicken. In this study, we investigated distribution of CAV antibody by ELISA and the virus gene by PCR in poultry farms in Jeongeup, Jeonbuk province. In the test using ELISA kit, 41 (95.3%) of 43 flocks and 88.6% of the individual chickens were positive, respectively. By PCR, 90.9% of the broiler breeders and 75.0% of White-semi breeders were found positive, respectively. All hatchery was negative by PCR. Of the clinical cases from 49 poultry flocks, 87.5% of flocks and 54.7% for each samples were found positive by ELISA, respectively. By PCR test, 21 (42.9%) of 49 flocks were positive. Major clinical signs of the infected flocks were growth retardation, femoral subcutaneous bleeding, depression, limping, and continuing selection. The genetic analysis of separate N genes of CAV showed highly homologous each other. The nucleotide sequence of field isolates had homology ranged from 99.9% to 97.5% with Chinese strains, and 99.9% to 99.6% with Japanese strain. Phylogenetic analysis based on the N gene of CAV isolates showed the closely relation with Chinese strains. The results of this survey could be used as basic data for development of vaccine.

• Journal of Acupuncture Research
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• v.23 no.2
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• pp.173-179
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• 2006

Objectives : In this study, we investigated the SNP (single-nucleotide polymorphism) of IL10 in patients with stroke. The present study was undertaken to see if specific genotypic and allelic variations are associated with stroke in the Korean population. Methods : Blood samples from all subjects were obtained for DNA extraction and collected in EDTA tube. Genomic DNA was extracted using DNA isolation kit for Mammalian Blood (Boehringer Mannheim, IN, USA). The extracted DNA was amplified by polymerase chain reaction (PCR). Pyrosequencing was performed according to manufacturer's standard protocol. Results : There was no statistically significant genotypic distribution difference between control and stroke group. The frequencies of A/A homozygotes and A/C heterozygotes among control subjects were 91 (87.5%) and 13 (12.5%). The frequencies of A/A and A/C among the stroke patients were 85 (89.5%) and 10 (10.5%). There was not statistically significant allelic frequency difference between control and stroke group. The allelic frequency of A and C was 195 (93.8%) and 13 (6.2%) among the control subjects and 180 (94.7%) and 10 (5.3%) in stroke patients, respectively. Conclusion : The cytokine IL10 may not be pathogenetic factors in stroke. But further studies including different cytokine gene can be a useful for predicting stroke. Establishment of more systemic approach and high quality of prospective cohorts will be necessary for the good prediction of genetic markers.