• Applied Biological Chemistry
• /
• v.38 no.5
• /
• pp.382-386
• /
• 1995

The potato proteinase inhibitor II (PI-II) protein contains chymotrypsin and trypsin inhibitory site. Among several PI-II genes isolated from genomic library, amino acid sequence deduced from PI-IIT gene has 84% identity with that of the polypeptide chymotrypsin inhibitor (PCI). Therefore a gene fragment having homology with the PCI was cloned into a vector using polymerase chain reaction(PCR) from the potato proteinase inhibitor IIT gene. Two different primers were utilized for cloning; primer A contains NdeI restriction site and 30 nucleotides, which has AUG N-terminal methionine codon, primer B contains BclI restriction site and 28 nucleotides, which has TAG translation stop codon. After PCR, about 160 bp-long DNA fragment was cloned into pRT146, derivative of pUC118, and sequenced. The sequenced NdeI/BclI fragment was moved to pET3a, containing bacteriophage T7 promoter and terminator. The expressed proteins in E. coli BL2l(DE3) were determined on a polyacrylamide gel containing sodium dodecyl sulfate. The expected size of protein deduced from the sequenced gene fragment is about 6,500 dalton whose size was similar to the IPTG-induced protein (6,000 dalton) on a gel. However the expression level was much lower than expected.

• Korean journal of applied entomology
• /
• v.53 no.1
• /
• pp.85-91
• /
• 2014

In this study, we analyzed the phylogenetic characterization of root-knot nematodes (Meloidogyne spp.) in soils from fruits and vegetables greenhouses in Korea. Soil samples were collected from 12 greenhouse fields in which tomato, cucumber, watermelon, and Oriental melon were being cultivated. Meloidogyne spp. were detected in all the soil samples at an average number of $72{\pm}6$ nematodes/300 g of soil to $2,898{\pm}468$ nematodes/300 g of soil. Phylogenetic analysis using polymerase chain reaction-restriction fragment length polymorphism was attempted for the second-stage juveniles (J2) of Meloidogyne spp. collected from the greenhouse soils. Twelve Meloidogyne spp. from the greenhouse soils were classified into two groups by using HinfI digestion of mitochondrial DNA, resulting in 900, 410, 290, and 170 bp fragments (group A) and 900, 700, and 170 bp fragments (group B). Phylogenetic analysis based on mitochondrial DNA sequences (1,483-1,521 bp) showed that nine group A isolates were identified as Meloidogyne incognita (99.73-99.93%) and three group B isolates showed 99.54-99.73% similarity to Meloidogyne arenaria.

• Korean Journal of Veterinary Service
• /
• v.31 no.1
• /
• pp.1-16
• /
• 2008

Salmonella infections cause the disease in pigs but also some zoonotic Salmonella serotypes can be transmitted to human through swine products, resulting in food poisoning. The objective of this study was to investigate the bacteriological prevalence and detection of invA gene using Salmonella specific polymerase chain reaction (PCR), the epidemiological characteristics related to Salmonella strains cultured from pig samples in Gangwon areas using serotyping, random amplified polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE) methods. During the period of November 2001 through April 2002, 1,174 ileocecal lymph node were collected from the slaughtered pigs raised in 38 farms located in Gangwon province. The samples were submerged in boiling water and macerated in saline and lymph node homogenates were inoculated into Tetrathionate broth with iodine (TTB, Difco, 0.5% iodine was added) for enrichment growth. Then additional tests were performed using several mediums, and suspects were identified by API 20E kit (BioMerieux) and PCR. Of total 1,174 samples from 38 farms, 44 (3.7%) were isolated as Salmonella spp from 13 farms (34.2%). Of 44 isolates, 31 were in Yangyang region, followed by 9 in Goseong, 2 in both Gangneung and Sokcho. However, there was no difference in regional isolation frequency. All isolates have a 521bp amplified product in Salmonella specific PCR with primer invA which encodes in proteins for invasion of epithelial cells. Of 44 recovered serotypes, 23 (52.3%) were S Eingedi, 10 (22.7%) S Schwarzengrund, 9 (20.5%) S Typhimurium, and 2 (4.5%) S Mbandaka. In RAPD analysis, there appeared to be unique bands distinguishing each serotype, although similarities exist between the different serotypes. Four serotypes of 44 Salmonella isolates appeared to fall into 14 different RAPD types. In PFGE analysis, 9 S Typhimurium were tested with XbaI enzyme and SpeI enzyme. The combination of results obtained with two enzymes subdivided the 9 S Typhimurium into 4 PFGE types.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.28 no.2
• /
• pp.71-84
• /
• 2014

The full-length heat shock protein 88 (HSP88) complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening the Paecilomyces tenuipes (P. tenuipes) Jocheon-1 Uni-Zap cDNA library and performing 5' RACE polymerase chain reaction (PCR). The P. tenuipes Jocheon-1 HSP88 cDNA contained an open reading frame (ORF) of 2,139-basepair encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipe s Jocheon-1 HSP88 cDNA showed 77% identity to Nectria haematococca HSP88 and 45-76% identity to other fungal homologous HSP88s. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipes Jocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade. The P. tenuipes Jocheon-1 HSP88 also contained the conserved ATPase domain at the N-terminal region. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as an 88 kilodalton (kDa) polypeptide in baculovirus-infected insect Sf9 cells. Under higher temperature conditions for the growth of the entomopathogenic fungus, mRNA expression of P. tenuipes Jocheon-1 HSP88 was quantified by real time PCR (qPCR). The results showed that heat shock stress induced a higher level of mRNA expression compared to normal growth conditions.

• Journal of Periodontal and Implant Science
• /
• v.35 no.3
• /
• pp.623-634
• /
• 2005

Human periodontal ligament fibroblast(hPDLF) is very important to cure periodontal tissue because it can be diverged into various cells. This study examined the expression of MMP-1, TIMP-1, periodontal ligament specific PDLs22, Type I collagen, Fibronectin, TIMP-2, telomerase mRNA in a replicative senescence of hPDLF. The periodontal ligament tissue was obtained from periodontally healthy and non-carious human teeth extracted for orthodontic reasons at the Chosun University Hospital of Dentistry with the donors' informed consent. The hPDLF cells were cultured in a medium containing Dulbecco's modified Eagle medium(DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, Gibco BRL, USA) at 37C in humidified air with 5% $CO_2$. For the reverse transcription-polymerase chain reaction(RT-PCR) analysis, the total RNA of the 2, 4, 8, 16, 18, and 21 passage cells was extracted using a Trizol Reagent(Invitrogen, USA) in replicative hPDL cells. Two passage cells, i.e. young cells, served as the control, and ${\beta}-actin$ served as the internal control for RT-PCR The results of this study about cell morphology and gene expression according to aging of hPDLF using RT-PCR method are as follows: 1. The size of hPDLF was increased with aging and it was showed that the hPDLF was dying in the final passage. 2. PDLs22 mRNA was expressed in young hPDLF of the two, four, and six passage. 3. TIMP-1 mRNA was expressed in young hPDLF of the two and four passage. 4. There was a tendency that MMP-1 mRNA was weakly expressed over eighteen. 5. Type 1 collagen mRNA was expressed in almost all passages, but it was not expressed in the final passage. 6. Fibronectin mRNA was observed in all passages and it was weakly expressed in the final passage. 7. TIMP-2 and telomerase mRNA were not expressed in this study. Based on above results, it was observed that PDLs22, Type 1 collagen, Fibronectin, MMP-1. and TIMP-1 mRNA in hPDLF were expressed differently with aging. The study using the hPDLF that is collected from healthy patients and periodontitis patients needs in further study.

• Korean Journal of Clinical Laboratory Science
• /
• v.41 no.4
• /
• pp.145-152
• /
• 2009

Novel influenza A virus, subtype H1N1 of swine-lineage, has been transmitted rapidly to many regions of the world. Rapid detection of the virus is essential to instigate appropriate patient care and public health management and for disease surveillance. The aim of this study is to determine the prevalence of novel influenza A (H1N1) virus in Korea using reverse-transcription real time polymerase chain reaction (rRT-PCR). Novel H1N1 virus was detected in a total of 8,948 nasopharyngeal samples from patients with influenza-like illness throughout Korea from August to September 2009. RNA was extracted from $300{\mu}l$of sample using an RNA extraction kit (Zymo Research, CA, USA). In the present study, Genekam kit (Genekam, Duisburg, Germany) was used to detect novel H1N1 virus. Novel H1N1 virus was found in 1,130 samples from a total of 8,948 samples (12.6%). The highest frequency was found in 10- to 19-year-olds (M: 29.3% vs. F: 16.4%), followed by 20- to 29-year-olds (M: 17.9% vs. F: 15.4%), 40- to 49-year-olds (M: 6.5% vs. F: 8.1%), 50- to 59-year-olds (M: 6.0% vs. F: 5.5%), and 30- to 39-year-olds (M: 4.6% vs. F: 3.8%). The mean positive rate was higher in men than in women (M: 14.7% vs. F: 7.4%). Novel H1N1 virus showed the lowest prevalence in patients over 60 years old. The positive rate increased daily and showed a significant high peak in mid-September 2009. In 19 provinces of Korea, Cheonan (41.1%), Busan (37.3%), Gangneung (33.3%), Jinju (32.1%), Ulsan (24.6%), Deajeon (23.7%) areas showed high frequencies and other provinces were found less than 10% of novel H1N1 virus. Since reverse-transcription real time PCR assay is rapid, accurate, and convenient, it may assist public health laboratories in detecting novel H1N1 virus. Moreover, these data could be useful for the management of patients with influenza-like illness.

• Korean Journal of Veterinary Research
• /
• v.60 no.2
• /
• pp.61-68
• /
• 2020

The zoonotic transmission of viral diseases to humans is a serious public health concern. Pigs are frequently a major reservoir for several zoonotic viral diseases. Therefore, periodic surveillance is needed to determine the infection rates of zoonotic diseases in domestic pigs. Hepatitis E virus (HEV), rotavirus, sapovirus (SaV), and norovirus (NoV) are potential zoonotic viruses. In this study, 296 fecal samples were collected from weaned piglets and growing pigs in 13 swine farms, and the viral RNA was extracted. Partial viral genomes were amplified by reverse transcription-polymerase chain reaction (PCR) or nested-PCR using virus-specific primer sets under different PCR conditions. HEV-3, rotavirus A, and SaV genogoup 3 were detected from 11.5, 2.7, and 3.0% of the samples, respectively. On the other hand, NoV was not detected in any of the samples. Genetic analysis indicated that the nucleotide sequences of swine HEV-3 and rotavirus A detected in this study were closely related to those of human isolates. However, swine SaV was distant from the human strains. These results suggest that HEV-3 and rotavirus A can be transmitted from pigs to humans. Therefore, strict preventive measures should be implemented by workers in the swine industry to prevent infections with HEV-3 and rotavirus A excreted from pigs.

• Korean Journal of Veterinary Service
• /
• v.25 no.3
• /
• pp.245-257
• /
• 2002

The gene encoding the HN protein from the CBP-1 strain, a heat stable Newcastle disease virus (NDV) isolated from diseased pheasants in Korea, was characterized by reverse transcriptase- polymerase chain reaction(RT-PCR) and the nucleotide and amino acid sequences were analyzed following cloning of the HN gene. In all of the NDV strains studied, a 1.75 kb size cDNA fragment for the HN gene was generated by RT-PCR and smaller specific band sizes harboring the internal portions of the HN gene were also detected by using four pairs of primers. The RT-PCR was sensitive enough to detect viral transcripts when the virus titer was above 25 hemagglutination units. The amplified 1.75 kb cDNA was cloned into a BamHI site of the pVL1393 Baculo transfer vector. The nucleotide sequences of the 1,758 bp HN gene from the CBP-1 strain were determined by the dye terminator cyclic sequencing method. The gene sequences were compared among the strains of CBP-1, Texas GB, Beaudette C, LaSota, B1 and Ulster. The homology of the CBP-1 HN gene to other HN variants was 97.8% to Texas GB, 98.4% to Beaudette C, 95.4% to LaSota, 95.6% to B1 and 90.2% to Ulster. As the deduced 577 amino acid sequences were compared among the strains, the homology for CBP-1 HN appeared to be 96.7% to Texas GB, 97.9% to Beaudette C, 95.5% to LaSota, 95.5% to B1 and 92.7% to Ulster. It was evident that the amino acid sequences included 5 sites for N-asparagine linked glycosylation and 12 cysteine residues. The three conserved leucine residues within the predicted transmembrane domain of the HN protein are amino acid 30, 37 and 44. The three antigenic sites on the HN protein of NDV are amino acids 347(Glu), 481(Asn) and 495(Glu). These data indicate that the genotype of the CBP-1 strain is more closely associated with the strains of Texas GB and Beaudette C than it is for the LaSota, B1 and Ulster strains.

• ALGAE
• /
• v.35 no.1
• /
• pp.45-59
• /
• 2020

To investigate the spatio-temporal distributions of the mixotrophic dinoflagellate Yihiella yeosuensis in Korean coastal waters and its grazing impact on prey populations, water samples were seasonally collected from 28 stations in the East, West, and South Seas of Korea and Jeju Island from April 2015 to October 2018. The abundances of Y. yeosuensis in the water samples were quantified using quantitative real-time polymerase chain reaction (qPCR). Simultaneously, the physical and chemical properties of water from all sampled stations were determined, and the abundances of the optimal prey species of Y. yeosuensis, the prasinophyte Pyramimonas sp. and the cryptophyte Teleaulax amphioxeia, were quantified using qPCR. Y. yeosuensis has a wide distribution, as is reflected by the detection of Y. yeosuensis cells at 23 sampling stations; however, this distribution has a strong seasonality, which is indicated by its detection at 22 stations in summer but only one station in winter. The abundance of Y. yeosuensis was significantly and positively correlated with those of Pyramimonas sp. and T. amphioxeia, as well as with water temperature. The highest abundance of Y. yeosuensis was 48.5 cells mL^-1 in Buan in July 2017, when the abundances of Pyramimonas sp. and T. amphioxeia were 917.6 and 210.4 cells mL^-1, respectively. The growth rate of Y. yeosuensis on Pyramimonas sp., calculated by interpolating the growth rates at the same abundance, was 0.49 d^-1, which is 37% of the maximum growth rate of Y. yeosuensis on Pyramimonas sp. obtained in the laboratory. Therefore, the field abundance of Pyramimonas sp. obtained in the present study can support a moderate positive growth of Y. yeosuensis. The maximum grazing coefficient for Y. yeosuensis on the co-occurring Pyramimonas sp. was 0.42 d^-1, indicating that 35% of the Pyramimonas sp. population were consumed in 1 d. Therefore, the spatio-temporal distribution of Y. yeosuensis in Korean coastal waters may be affected by those of the optimal prey species and water temperature. Moreover, Y. yeosuensis may potentially have considerable grazing impacts on populations of Pyramimonas sp.

• Pediatric Infection and Vaccine
• /
• v.18 no.2
• /
• pp.173-181
• /
• 2011

Purpose : 2009 Pandemic influenza A (H1N1) virus was identified in March 2009 and subsequently caused worldwide outbreaks. We described the clinical and epidemiological characteristics of H1N1 influenza infection. Methods : We used retrospective medical chart reviews to collect data on the visiting patients from a single institute. H1N1 infection was confirmed in specimens with the use of a RT-PCR (real time reverse transcriptase polymerase chain reaction assay). Result : 6,836 patients had H1N1 RT-PCR test, and 2,781 were confirmed with H1N1 virus infection. 158 patients (5.7%) had hospital treatment and inpatients were significantly younger (5.4${\pm}$3.3 years) than outpatients (7.5${\pm}$3.9 years) among H1N1 virus confirmed patients. Oxygen, steroid, immunoglobulin, ventilator treatment was provided in a substantial proportion among pneumonia patients accompanying wheezy respiration. In addition more intensive care was needed in patients accompanying segmental, lobar, interstitial, mixed pneumonia and lung effusion (27.2%) than patients with bronchopneumonia (7.3%) among H1N1 virus infection confirmed patients. Seventy-one infants had oseltamivir treatment out of 83 infants under 1 year, and no significant side effects and complications were identified. Conclusion : In 2009 pandemic influenza A (H1N1), hospital treatment was needed in younger patients. Early intensive care was needed in pneumonia patients accompanying wheezy respiration, and patients accompanying segmental, lobar, interstitial, mixed pneumonia and lung effusion.