• IMMUNE NETWORK
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• v.3 no.2
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• pp.126-135
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• 2003

Background: One of the important factors in the prognosis of chronic hepatitis B patient is the degree of replication of hepatitis B virus (HBV). It has been known that HBV DNA polymerase plays the essential role in the replication of HBV. HBV DNA polymerase is composed of four domains, TP (Terminal protein), spacer, RT (Reverse transcriptase) and RNaseH. Among these domains, tyrosine, the $65^{th}$ residue of TP is an important residue in protein-priming reaction that initiates reverse transcription. If monoclonal antibody that recognizes around tyrosine residue were selected, it could be applied to further study of HBV replication. Methods: To produce TP-specific scFv (single-chain Fv) by phage display, mice were immunized using synthetic TP-peptide contains $57{\sim}80^{th}$ amino acid residues of TP domain. After isolation of mRNA of heavy-variable region ($V_H$) and light-chain variable region ($V_L$) from the spleen of the immunized mouse, DNA of $V_H$ and $V_L$ were obtained by RT-PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a scFv DNA fragments. ScFv DNA fragments were cloned into a phagemid vector. scFv was expressed in E.coli TG1 as a fusion protein with E tag and phage gIII. To select the scFv that has specific affinity to TP-peptide from the phage-antibody library, we used two cycles of panning and colony lift assay. Results: The TP-peptide-specific scFv was isolated by selection process using TP-peptide as an antigen. Selected scFv had 30 kDa of protein size and its nucleotide sequences were analyzed. Indirect- and competitive-ELISA revealed that the selected scFv specifically recognized both TP-peptide and the HBV DNA polymerase. Conclusion: The scFv that recognizes the TP domain of the HBV DNA polymerase was isolated by phage display.

• Korean Journal of Veterinary Research
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• v.41 no.1
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• pp.89-98
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• 2001

This study was conducted to detect the toxoplasma specific-DNA in circulating blood and organs collected from slaughtered pigs at slaughtering house and experimentally infected pigs with Toxoplasma gondii tachyzoites by polymerase chain reaction(PCR), and also PCR was applied to diagnose for acute phase of swine toxoplasmosis as a newly developed diagnostic test. The sensitivity of oligonucleotide primer, T-1 & T-2, designed from toxoplasma B1 gene amplification method was compared with Tp parasite detection by mouse inoculation(MI). On the other hand, latex agglutination test(LAT) was conducted to detect the serum antibodies comparing with the detection of toxoplasma by PCR and MI. The results obtained were summarized as follows. PCR was able to determine at the lowest level of $10^0/ml$ T. gondii in blood samples which were blended with a serial diluted T gondii in vitro. On the other hand, $10^2/5g$ of T gondii could detect from a variety of tissues including lung, diaphragm, liver, heart, spleen and brain in vitro. The primer was proved to specifically determine T gondii in blood and tissues in vitro but it did not detect Neospora caninum used as a negative control. DNA of T. gondii was effectively extracted by freezing, thawing and grinding twice both tissues mixed with T gondii in vitro and in experimentally infected pig's tissues. PCR detected specific DNA in the blood of experimentally infected pigs at 108 hrs and 120 hrs post-infection, it was the same time that the pigs showed fever and parasitaemia. In case of tissue, specific DNA was, however, detected only lung from experimentally infected pigs. Even though the duration of acute phase was from 3 to 7 days post-infection, but the latex agglutination test (LAT) results appeared from 8 days post-infection. A comparison of sensitivity in determining T gondii in blood samples between PCR and MI, PCR positive rate ranged from 25 to 33.3%, but that of MI covered from 75 to 100%.

• Journal of Plant Biotechnology
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• v.7 no.1
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• pp.51-55
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• 2005

Many cloning vectors in which cDNAs can be inserted to the sense orientation have been developed. Uni-ZAP XR vector (Stratagene) should contain clones that are oriented to sense direction with respect to T3 RNA polymerase primer. Unexpectedly, large portions of cDNAs in Chinese cabbage cDNA library showed unusual insertions, antisense orientation and a hybrid of two different clones. Using two clones, 4H03 and 53-B10, derived from different cDNA libraries, we proposed and demonstrated the possibility of unusual-construct formation by in vitro translation and northern blot analysis. The 4H03 clone was inserted with inverse direction, and its transcript and translation product could be produced by T7 RNA polymerase, indicating that this clone is definitely inserted into inverse orientation. The 53-B10 that contains two independent genes was turned out to be a hybrid in which two genes are inserted to opposite direction each other. All unusual constructs might be due to the presence of small fragments of DNA, like adapter. However, the mechanism underlined the formation of unusual constructs is still remain to be solved.

• Biomedical Science Letters
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• v.4 no.2
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• pp.113-120
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• 1998

To investigate the distribution of Borrelia burgdorferi and human granulocytic ehrlichiosis (HGE) agent in ticks, adult ixodid ticks of Ixodes spp. and Haemaphysalis spp. were collected from the high mountain areas of Kangwon Province. Using DNAs extracted and purified in the collected ticks, polymerase chain reaction (PCR) was performed to amplify the specific nucleotide sequences of both agents. Of the 516 ticks, a total of 68 (13.2%) ticks was positive for Borrelia burgdorferi sensu lato with PCR analysis (2 for B. burgdorferi sensu stricto； 1 for B. afzelii；33 for B. garinii； 8 for B. tanukii；4 for B. turdae). However a little more than half of PCR-positive ticks (37/68) was found to be positive in the southern blot analysis with Bl6S oligonucleotide probe. One hundred and one (19.2%) ticks were positive for Ehrlichia spp. in PCR, and a quarter of them (25/101) was positive in southern blot with El6S oligonucleotide probe. But none of them was found to be the DNA of HGE agent. And 0.6% (3/516) ticks were positive for both of B. burgdorferi sensu late and Ehrlirhia spp. These findings might implicate the possibility of the outbreak of Iyme borreliosis and ehrlichiosis in Korea, and more extensive studies may be need for the diagnosis of multiple tick-borne diseases.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2012.05a
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• pp.913-915
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• 2012

RNA polymerase beta subunit gene (rpoB) mutation of rifampin-resistant Mycobacteria was analyzed using nucleotide sequence of rpoB DNA (351 bp) containing rifampin resistant region, $rif^r$. For this purpose, we collected rifampin-resistant Mycobacteria that were identified by conventional culture method from Masan National Hospital and The Korean Institute of Tuberculosis and performed analysis of nucleotide sequence of rpoB of them. We found various mutations of rpoB linked rifampin resistant gene from rifampin-resistant Mycobacteria. From this study, we identified mutations of different codons from codons that have been reported recently.

• Tuberculosis and Respiratory Diseases
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• v.39 no.3
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• pp.242-247
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• 1992

Background: The ability to precisely measure specific mRNA levels by hybridization to complementary DNA probes is an important tool for analyzing the regulation of gene expression. Surfactant proteins have important roles in regulating surfactant metabolism as well as in determing its physical properties. Method: The complete coding regions for rat surfactant protein complementary DNA of surfactant protein B were subcloned into pGem 3Z or 4Z such that either antisense or sense transcripts were obtained by using SP 6 RNA polymerase. Surfactant protein B mRNA was measured by filter hybridization. Results: Equation of standard curve between counts per minute (Y) and surfactant protein B mRNA transcript input (X) was Y=2034.9 X+159.1. Correlation coefficient was 1.0. Couclusions: Filter hybridization assay is suited to situation when rapid, accurate quantitation of multiple samples is required.

• Korean Journal of Clinical Laboratory Science
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• v.39 no.3
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• pp.147-150
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• 2007

Most expressed HLA (human leukocyte antigen) loci exhibit a remarkable degree of allelic polymorphism, which derives from sequence differences predominantly localized to discrete hypervariable regions of the amino terminal domain of the molecule. In this study, the HLA-B genotypes were determined in twenty students unrelated koreans using the PCR-SSP (polymerase chain reaction-sequence specific primer) technique. Several specific primer pairs in assigning the HLA-B gene were used ($B^{\ast}4001/4007$, $B^{\ast}4901/5001/4501$, $B^{\ast}3701$, $B^{\ast}5801$). The results of PCR-SSP, the HLA-B3701 primer was detected one (5%), the $HLA-B^{\ast}5801$ were detected four (20%), the $HLA-B^{\ast}4001/4007$ were detected nineteen (95%) and the $HLA-B^{\ast}4901/5001/4501$ were detected twenty. This study shows that the PCR-SSP technique is relatively simple, fast and a practical tool for the determination of the HLA-B genotypes. Moreover, these results genotype frequency of the HLA-B gene could be useful for database study before being applied to individual identification and transplantation immunity.

• Proceedings of the Korean Society of Toxicology Conference
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• 2001.10a
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• pp.9-9
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• 2001

Several micronutrients (vitamins and minerals) are required as co-factors in DNA synthesis, DNA repair, DNA methylation and apoptosis. Some notable examples include (a) folic acid and vitamin B12 required for maintenance methylation of DNA and the synthesis of dTTP from dUTP, thus prevent the misincorporation of uracil into DNA, a highly mutagenic and chromosome-breaking event, (b) niacin, is essential in the form of the coenzymes NAD and NADP which act as a substrate for polyADPribose polymerase (PARP), an enzyme thought to facilitate efficient DNA repair and telomere length regulation and (c) zinc, apart from its antioxidant role as a co-factor in Cu/Zn SOD, it is required in its stabilizing role of the DNA-binding domain of p53 (residues 102-292) and thus is essential for apoptotic response to DNA damage. (omitted)

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.16-24
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• 2021

Hepatitis B virus (HBV) genome P-encoded protein HBV DNA polymerase (Pol) has long been known as a reverse transcriptase during HBV replication. In this study, we investigated the impact of HBV Pol on host cellular processes, mainly apoptosis, and the underlying mechanisms. We showed a marked reduction in apoptotic rates in the HBV Pol-expressed HepG2 cells compared to controls. Moreover, a series of assays, i.e., yeast two-hybrid, GST pull-down, co-immunoprecipitation, and confocal laser scanning microscopy, identified the host factor eEF1A2 to be associated with HBV Pol. Furthermore, knockdown of eEF1A2 gene by siRNA abrogated the HBV Pol-mediated anti-apoptotic effect with apoptosis induced by endoplasmatic reticulum (ER) stress-inducer thapsigargin (TG), thus suggesting that the host factor eEF1A2 is essential for HBV Pol's anti-apoptosis properties. Our findings have revealed a novel role for HBV Pol in its modulation of apoptosis through integrating with eEF1A2.

• The Korean Journal of Nuclear Medicine Technology
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• v.23 no.1
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• pp.35-39
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• 2019

Purpose Hepatitis B virus (hepatitis B virus, HBV) infection is a worldwide major public health problem and it is known as a major cause of chronic hepatitis, liver cirrhosis and liver cancer. And serologic tests of hepatitis B virus is essential for diagnosing and treating these diseases. In addition, with the development of molecular diagnostics, the detection of HBV DNA in serum diagnoses HBV infection and is recognized as an important indicator for the antiviral agent treatment response assessment. We performed HBeAg assay using Immunoradiometric assay (IRMA) and Chemiluminescent Microparticle Immunoassay (CMIA) in hepatitis B patients treated with antiviral agents. The detection rate of HBV DNA in serum was measured and compared by RT-PCR (Real Time - Polymerase Chain Reaction) method Materials and Methods HBeAg serum examination and HBV DNA quantification test were conducted on 270 hepatitis B patients undergoing anti-virus treatment after diagnosis of hepatitis B virus infection. Two serologic tests (IRMA, CMIA) with different detection principles were applied for the HBeAg serum test. Serum HBV DNA was quantitatively measured by real-time polymerase chain reaction (RT-PCR) using the Abbott m2000 System. Results The detection rate of HBeAg was 24.1% (65/270) for IRMA and 82.2% (222/270) for CMIA. Detection rate of serum HBV DNA by real-time RT-PCR is 29.3% (79/270). The measured amount of serum HBV DNA concentration is $4.8{\times}10^7{\pm}1.9{\times}10^8IU/mL$($mean{\pm}SD$). The minimum value is 16IU/mL, the maximum value is $1.0{\times}10^9IU/mL$, and the reference value for quantitative detection limit is 15IU/mL. The detection rates and concentrations of HBV DNA by group according to the results of HBeAg serological (IRMA, CMIA)tests were as follows. 1) Group I (IRMA negative, CMIA positive, N = 169), HBV DNA detection rate of 17.7% (30/169), $6.8{\times}10^5{\pm}1.9{\times}10^6IU/mL$ 2) Group II (IRMA positive, CMIA positive, N = 53), HBV DNA detection rate 62.3% (33/53), $1.1{\times}10^8{\pm}2.8{\times}10^8IU/mL$ 3) Group III (IRMA negative, CMIA negative, N = 36), HBV DNA detection rate 36.1% (13/36), $3.0{\times}10^5{\pm}1.1{\times}10^6IU/mL$ 4) Group IV(IRMA positive, CMIA negative, N = 12), HBV DNA detection rate 25% (3/12), $1.3{\times}10^3{\pm}1.1{\times}10^3IU/mL$ Conclusion HBeAg detection rate according to the serological test showed a large difference. This difference is considered for a number of reasons such as characteristics of the Ab used for assay kit and epitope, HBV of genotype. Detection rate and the concentration of the group-specific HBV DNA classified serologic results confirmed the high detection rate and the concentration in Group II (IRMA-positive, CMIA positive, N = 53).