DNA중합효소 연쇄반응을 이용한 한국형 젖소 면역결핍 바이러스의 검출

권오식 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1999 Journal of biomedical laboratory sciences Vol.5 No.1

젖소 면역결핍 바이러스 (BIV)는 젖소에게 여러 가지 면역결핍증후군을 야기하는 렌티바이러스 (역전사효소 바이러스의 아군)로서 국내에서는 이에 대한 연구나 보고가 진행된 바 없다. 본 연구에서는 BIV가 다른 젖소의 역전사효소 바이러스인 젖소 백혈병바이러스 (BLV)와 동시 감염되고 (5% 정도)있는 점을 착안해 우선 대구·경북지역의 소의 BLV 감염실태를 조사해 BLV 가 감염된 젖소를 대상으로 BIV 감염상태를 알아보았다. 먼저 10회에 걸쳐 젖소와 황소 248마리의 혈액을 채취해 BLV와 BIV의 숙주세포가 되는 말초혈액 단핵구 (peripheral blood monocytes, PBMC)를 분리하고, 이를 이용해 DNA 중합효소 연쇄반응 (PCR)과 Southern blot 분석법을 통해 BLV와 BIV의 존재여부를 조사하였다. 그 결과 조사대상 젖소의 66.9%(81/121) 이상이 BLV에 감염되어 있음을 PCR 검사를 통해 알 수 있었으며, 이는 Southern blot법으로 재차 확인되었다. 이 결과는 종래에 보고된 대구·경북지역에서의 BLV 감염율인 27∼30%보다 2배 이상 높은 수치로서, (1) 우리가 사용한 방법들 (PCR & Southern blot법)이 분자생물학적 연구방법인 관계로 고도의 특이성 (specificity)을 지녔기 때문이며 (2) 지난 10년간 조사 대상지역인 대구·경북지역 내에서 젖소들에게 지속적으로 BLV 감염이 증가하였기 때문으로 본다. 한편 이들 BLV positive PBMC의 chromosomal DNA를 사용해 BIV의 검출을 시도한 바, lot 3C 샘플은 BLV에 100% 감염되어 있음과 동시에 그 일부가 BIV에 감염되어 있음을 PCR 방법을 통하여 확인할 수 있었다. Bovine immunodeficiency virus (BIV) which was grouped into the Lentivirinae of family Retroviridae, was known to be causing many immunodeficiency syndromes among cows. The BIV was studied worldwide during last several years for its importance in cattle industries but nothing was reported in Korea until now. Thus we initially tried to study the existence of BIV in cattle around the Daegu·Kyungpook area by PCR related molecular techniques. As a prerequisite investigation for detecting Korean-type BIV, we had focused our aim into BLV infected cows because the BLV infected cows tend to show BIV infection with 5% ranges. Hence we randomly sampled fresh bloods from 248 cows and bulls near the Daegu·Kyungpook area and collected peripheral blood monocytes (PBMC) from the sample bloods. After extracting genomic DNA from the PBMC, we subjected it to PCR and Southern blot analysis for BIV/BLV detection. Overall, 66.9% (81/121) of the cow PBMC samples turned out to be BLV positive by PCR and the result was reconfirmed by Southern blot analysis. The value was two times higher than the previously reported results of BLV infection in Korea. The significant difference was mainly due to 1) applying highly specific methods for BLV detection such as PCR 2) that BLV was continuously spreaded in the Daegu·Kyungpook area without any notice during last ten years. We also tested the BLV positive samples with the same techniques for BIV detection. And we found some BIV positives among the lot 3C samples by PCR, which had showed 100% BLV positive.

A Study on DNA Sequences and Mutation of Integrase Region of Korean-type Bovine Leukemia Virus (BLV) pol Gene

Kwon, Oh-Sik,Kang, Jung-Soon,Park, Hyun-Jin,Yoo, Min The Korean Society for Biomedical Laboratory Scien 2004 Journal of biomedical laboratory sciences Vol.10 No.1

Bovine leukemia virus (BLV) is a causative agent for lymphoma disease in cattle including cows worldwide. BLV shares similar virion structure and characteristics with other retroviruses. The pol gene of the BLV genome produced reverse transcriptase (RT) and integrase (IN) for important roles for BLV genome integration into host cell chromosomes that is known to be coded in the 3' side of the BLV pol gene (one third portion). In this study, we have sequenced 978 bp in the 3' side of the BLV pol gene from BLV 10C3 in order to determine the BLV IN region of it. And we compared it to the nucleotide sequences of an Australian BLV isolate. As a result, nucleotide sequences of the IN region of the Korean-type BLV pol gene were mutated at a rate of 3.7%. We can confirm that the typical mutations are such as Arg (AGG) $\rightarrow$ Lys (AAG), Thr (ACG) $\rightarrow$ Met (ATG), Ile (ATT) $\rightarrow$ Val (GTT), Asn (ACC) $\rightarrow$ His (CAC), Phe (TTT) $\rightarrow$ Leu (TTG) and Asn (ACC) $\rightarrow$ Asp (GAC). From the analysis of the sequencing data, we were able to determine the zinc-finger-like "HHCC" motif in the amino terminus of BLV IN, that was H-$X_3$-H-$X_{25}-C-X_2$-C. It was also found the DD35E motif in the IN catalytic domain as D-$X_{56}$-D-$X_{35}$-E. It fits very well to the consensus sequences of retroviral IN as well as HHCC motif.

A Study of genetic polymorphisms of HLA-class I and II Genes Using Polymerase Chain Reaction

Lee,Kyung-Ok THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1998 Journal of biomedical laboratory sciences Vol.4 No.1

HLA 유전자군은 인간의 유전자 중에서 가장 높은 유전적 다형성을 보이며, 분석방법에 따라 판정할 수 있는 대립유전자의 수에 많은 차이가 있다. 현재까지 혈청학적 방법을 이용하여 HLA 항원형 구분을 하였으나, 최근 골수이식 등 여러 HLA 활용분야에서 HLA 유전자형 분석이 요구되고 있어, 많은 수의 HLA 유전자형을 쉽고 정확하게 구분할 수 있은 HLA DNA typing 방법이 필요한 실정이다. 본 연구에서는 HLA-A, B, C, DRB1 유전자형 구분은 PCR SSP 방법을, HLA-DQA1, DQB1, DPB1 유전자형 구분은 PCR-RFLP 방법을 사용한 HLA DNA typing 방법으로 한국인에서 HLA유전자형을 구분하고자 하였다. 본 방법을 이용하여 HLA형이 규명된 B-임파아구 표준세포에서 DNA typing을 실시하였을 때, 11차 국제조직적합성학회에서 발표된 결과와 모두 일치하였다. 한국인에서 HLA-A, -B, -C,대립유전자는 17종, 23종, 16종이 확인되었으며, HLA-DQA1, -DQB1, -DPB1, DRB1대립유전자는 8종, 16종, 13종, 37종이 확인되었다. 한국인에서 빈도가 높은 HLA-class Ⅰ유전자는 HLA-A 유전자에서 A*02가 27.0%였으며 HLA-B 유전자에서는 B*40이 17.6%를 나타내었고 HLA-C 유전자에서는 Cw*0101이 19.2%로 가장 높은 빈도를 나타내었다. 한국인에서 가장 빈도가 높은 HLA-classⅡ 유전자는 DQA1 유전자에서 DQA1*0301이 32.1%였고, DQB1 유전자에서는 DQB1*0303이 12.9%를 나타내었으며, DPB1 유전자에서는 DPB1*0501이 31.3%였고 DRB1 유전자에서는 DRB1*1501이 9.2%를 나타내었다. 본 연구에서 실시한 HLA DNA typing 방법은 비교적 빠른 시간내에 많은 종류의 HLA 대립유전자형을 정확하게 구분할 수 있으므로 앞으로 tissue typing 실험실에서 유용하게 활용될 수 있을 것으로 생각된다. 또한 DNA typing 방법을 이용하여 분석한 한국인의 HLA-classⅠ,Ⅱ 유전자군의 유전자형빈도는 골수이식을 비롯한 각종 이식검사, 특수 질환 관련검사나 인류유전학연구 등 HLA 유전자의 임상적 활용을 위한 자료로 사용될 수 있을 것으로 기대된다. The HLA genes located in the short arm of chromosome 6 specify heterodimeric glycoproteins involved in the regulation of the immune response. Recently, in the elucidation of HLA polymorphism, serological and cellular typing methods have been replaced by DNA typing using polymerase chain reaction (PCR). The purpose of this study was to establish the HLA DNA typing methods and determine gene frequencies of HLA molecules in Koreans. PCR-SSP(sequence specific primers) and PCR-RFLP (restriction fragment length polymorphism) techniques were used for the analysis of HLA-A, -B, -C, DRB1 genes and HLA-DQA1, DQB1, DPB1 genes, respectively. The results of B-lymphoblastoid cells used for control experiment were consistent with the previous data identified in the 11th International Histocompatibility Workshop. Seventeen, 23, 16, 8, 16, 13 and 37 types of HLA-A, B, C, DQA1, DQB1, DPB1 and DRB1 alleles were found, respectively, in a total of unrelated 120 Korean individuals. The most frequent HLA alleles were A*02 (27.0%), B*40 (17.6%), Cw*01 (19.2%), DQA1*0301 (32.1%), DQB1*0303 (12.9%), DPB1*0501 (31.3%) and DRB1*1501 (9.2%) among Koreans. Theis study shows that DNA typing method using PCR technique is a relatively simple, fast and practical tool for the determination of the HLA-class Ⅰand Ⅱ genes. Moreover, the data of HLA gene frequencies could be useful for the Korean database before clinical applications, including organ and unrelated bone marrow transplantation, anthropological study, disease association and individual identification.

A Study on DNA Sequences and Mutation of Integrase Region of Korean-type Bovine Leukemia Virus (BLV) pol Gene

권오식,--,--,-- THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 2004 Journal of biomedical laboratory sciences Vol.10 No.1

Bovine leukemia virus (BLV) is a causative agent for lymphoma disease in cattle including cows worldwide. BLV shares similar virion structure and characteristics with other retroviruses. The pol gene of the BLV genome produced reverse transcriptase (RT) and integrase (IN) for important roles for BLV genome integration into host cell chromosomes that is known to be coded in the 3' side of the BLV pol gene (one third portion). In this study, we have sequenced 978 bp in the 3'side of the BLV pol gene from BLV 10C3 in order to determine the BLV IN region of it. And we compared it to the nucleotide sequences of an Australian BLV isolate. As a result, nucleotide sequences of the IN region of the Korean-type BLV pol gene were mutated at a rate of 3.7%. We can confirm that the typical mutations are such as Arg (AGG) → Lys(AAG), Thr (ACG) → Met (ATG), Ile (ATT) → Val (GTT), Asn (ACC) → His (CAC), Phe (TTT) → Leu (TTG) and Asn (ACC) → Asp (GAC). From the analysis of the sequencing data, we were able to determine the zinc-finger-like "HHCC" motif in the amino terminus of BLV IN, that was H-X_(3)-H-X_(25)-C-X_(2)-C. It was also found the DD35E motif in the IN catalytic domain as D-X_(56)-D-X_(35)-E. It fits very well to the consensus sequences of retroviral IN as well as HHCC motif.

HLA-B27 DNA Typing using Group Specific Polymerase Chain Reaction

Kim,Min-Jung,Lee,Kyung-Ok,Hong,Sung-Hoi,Oh,Moon-Ju,Kim,Kyung-In THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1996 Journal of biomedical laboratory sciences Vol.2 No.2

HLA-class I 항원의 HLA-B 유전자좌에 존재하는 HLA-B27 유전자는 임상적으로 강직성 척수염과 강한 관련성이 있음이 보고되고 있으며, 현재 HLA 유전자중 질병과의 관련성을 보기 위한 검사로 임상에서 가장 널리 사용되고 있다. 대부분의 검사실에서는 현재까지 혈청학적 검사방법을 이용하여 HLA-B27 검사를 실시하고 있는데, 이 방법은 시약이 고가이고, 검체의 안정성과 보관이 어려우며, 분석시간이 오래 걸리는 등 불편한 점이 있고, 또한 현재에도 계속 새로운 HLA-B27 대립유전자가 발견되고 있으므로 위음성의 가능성도 배제할 수 없어, 보다 정확한 검사방법이 요구되고 있다. 최근 HLA-B27 대립유전자의 염기배열이 대부분 밝혀져 혈청학적 방법 대신 DNA를 이용한 typing 방법이 보고되고 있다. 저자들은 HLA-B27 대립유전자에 공통으로 존재하는 염기배열 부분을 선택하여 group specific PCR(Polymerase Chain Reaction)을 실시하고 그 유용성을 검토하였다. 혈청학적 방법으로 HLA-B27 형이 확인된 검체 56 개와 4 개의 표준세포주(HOM-2, JESTHOM, WT24, BTB)를 이용하여 혈청학적 방법과 DNA typing을 비교한 결과, 두 방법사이에 완벽한 일치를 나타내었다. 따라서 group specific PCR을 이용한 HLA-B27 DNA typing은 검체 및 시약의 안정성이 높고, 경제적이며 신속한 검사가 가능하므로 임상에서 활용성이 매우 클 것으로 사료된다. HLA-B27 gene, one of the HLA-class I molecule, is strongly associated with ankylosing spondylitis. It has been most frequently used as a disease-correlated HLA gene by clinicians. In most laboratories, conventional HLA-B27 typing is still performed by cell cytotoxicity tests or fluorescence serology with specific antibodies. In this study, DNA typing method for HLA-B27 was developed by using group specific Polymerase Chain Reaction (PCR). Four HLA­B27 cell lines (HOM-2, JESTHOM, WT24 and BTB) and fifty six B27 Korean individuals defined by serology were used. The results of control cell and' B-27 positive individual samples were correlated well with the data which was performed by serological method. All of B27 positive PCR products gave positive signals on Southern blot hybridization with B27 specific probe. This study shows that the HLA-B27 DNA typing is a relatively simple, fast and practical tool for the determination of the HLA-B27 gene in routine clinical laboratory work.

Thermal Comport Condition of Temperature and Humidity in Loess Interior Space

공성훈 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 2004 Journal of biomedical laboratory sciences Vol.10 No.2

The study was carried out through measuring the temperature and humidity of the indoor/outdoor space and the distribution cf interior thermal condition, and investigating the effect of loess materials on human body. The purpose of this study is to analyze the change of dry bulb temperature and relative humidity and correlation of thermal reaction of human body with ASHRAE (American Society of Heating, Refrigerating and Air-conditioning) comfort chart in the loess interior space. In the view point of biomedical sciences, loess interior space provides optimum thermal conditions for human thermal sensation.

Genetic Polymorphisms of the Human Thyroid Peroxidase Gene Using Amplified Fragment Length Polymorphism : Application to the Determination of Paternityu in a Korean Polulation

Lee,Kyung-Ok,Kim,Yoon Jung,Oh,Moon-Ju,Kim,Eun-Ha,Lee,Kyu-Pum,Park,Taek-Kyu,Park,Young-Suk THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCINE 1995 Journal of biomedical laboratory sciences Vol.1 No.1

최근까지 개인식별검사는 주로 단백질의 다형성을 보는 것이었으나, 분자생물학의 발달로 개인식별 분야에도 DNA를 이용한 분석이 가능하게 되었다. 본 실험에서는 Thyroid Peroxidase(TPO)유전자의 다형성을 개인식별 및 친자감별검사에 이용하기 위하여 그 기초조사로 한국인에서 TPO유전자의 대립유전자 발현빈도와 돌연변이율 등을 포함한 평가실험을 실시하였다. 실험대상으로는 혈연관계가 없는 123명을 대상으로 말초혈액에서 DNA를 추출하고 PCR(중합효소연쇄반응)을 이용한 Amp-FLP방법을 이용하여 TPO 유전자를 증폭하였으며, polyacrylamide gel로 확인하였다. 그 결과 10개의 대립유전자와 29개의 유전형이 구분되었고, 이형접합성(Heterozygosity)은 70.7%였으며 돌연변이율은 0.075였고, 혈연 관계가 전혀 없는 두 사람이 동일한 유전자형을 가질 가능성(Probability)은 14.6×10-²이었다. 또한 x²=4.48, 0.05<P<1로써 Hardy-Weinberg법칙이 성립함을 보여주었다. 이상의 결과로 볼 때 TPO유전자는 한국인에서 개인식별이나 친자감별검사를 위한 유전적 표지자로 사용될 수 있을 것으로 사료된다. Genetic polymorphisms due to variation in the number of tandem repeats of DNA sequences(VNTRS) provides a useful means for discrimination between individuals. Allele and genotype frequencies of the highly polymorphic Human Thyroid Peroxidase(TPO) gene were determined in Korean population samples by using PCR followed by polyacrylamide gel electrophoresis, a procedure called the amplified fragment length polymorphism(Amp-FLP) technique. In 123 unrelated Korean individuals 10 different alleles and 29 genotypes were observed. The TPO gene demonstrated a heterozygosity of 0.707 and the power of exclusion(POE) was 0.945. The probability of having the same DNA band within two unrelated individuals was 14.6×10-². The distribution of observed genotypes conformed to Hardy-Weinberg equihbrium(x²=4.48, 0.05<P<0.1, df=2). In 30 paternity cases Mendelian inheritance for the alleles at the TPO gene could be demonstrated in all cases, and the mutation rate was 0.075.

Survery of Seroconversion Rates against Hantavirus in Laboratory Rodents

우영대,--,--,--,-- THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 2003 Journal of biomedical laboratory sciences Vol.9 No.2

Hantavirus ale rodent-borne RNA virus that belongs to the family Bunyaviridae. Those viruses persistently infect a variety of rodents, and are transmitted by aerosols of their urine, feces and saliva. Antibody titers of sera obtained from normal laboratory rodents against hantavirus were investigated by indirect immunofluorscence antibody technique (IFA). Seroconversion rates of normal laboratory rodents showed higher in rats than that from hamster and mongolian (M), gerbil. Theses rates of normal laboratory rodents also showed higher in titers against puumala virus (PUUV) than in hantaan (HTNV) and seoul virus (SEOV). We are concerned about infections caused by hantaviruses, especially by PUUV, occurred in laboratory rodents.

한국인 Fragile X 환자들의 혈청단백질 구성

김종봉 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1996 Journal of biomedical laboratory sciences Vol.2 No.1

한국인 일반 정신박약자들의 핵형 및 혈청단백질을 분석한 결과는 다음과 같다. 정신박약자 35명중 3명에서 fragile X 염색체를 발견하였고 그 빈도는 4∼15%였다. Fragile X 증후군 환자들의 혈청단백질의 농도는 5.73±0.89(g/dl)이었고 albumin과 globulin의 비는 0.86±0.14이었다. 일반 정신박약자들의 혈청단백질 농도는 6.83±0.72(g/dl)이었고 이중 albumin과 globulin의 비는 0.87±0.47이었다. Fragile X 증후군환자 및 일반정신 박약자들의 혈청단백질 농도 및 albumin과 globulin의 비는 정상인및 Down 증후군환자들 보다 낮았다. The karyotype and the concentration of serum protein were investigated in Korean unclassified mental retardees. The results were as follows. Fragile X chromosomes were identified in three patients, and the frequencies of fragile X chromosome were 4∼15%. The concentration of serum protein was 5.73±0.89(g/dl), and the A/G ratio was 0.86±0.14 in fragile X syndrome patients. The concentration of serum protein was 6.83±0.72(g/dl), and the A/G ratio was 0.87±0.17 unclassified mental retardees. The rusults revealed that the level of globulin concentration and A/G ratio in fragile X syndrome patients and unclassified mental retardees were lower than in normal group and Down's patients.

국내 Human Immunodeficiency Virus(HIV) 감염자와 정상인의 면역학적 표지인자 비교연구

신영오,류재천,최병선,박용근 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCINE 1995 Journal of biomedical laboratory sciences Vol.1 No.1

HIV감염자는 질병의 진전에 무관하게 감염 후의 경과시기에 따라서 CD4 T림프세포등 각종 면역상태를 나타내는 표지가 변한다. 따라서 HIV감염자의 질병진전을 예보하기 위하여서는 정기적으로 CD4등 각종표지를 측정하여 감염자의 질병상태를 monitoring하게 된다. 그러나 이러한 수치를 감염자관리에 적용하기 위하여서는 우리나라 일반인의 정상치를 파악하여 이를 지표로 해야하므로 국내정상인의 각종면역치에 대한 조사가 요구된다. 현재의 기준으로는 500이하로 떨어질 때에는 예방차원에서 AZT를 복용하게 되며 200이하로 떨어지면 질병의 유무에 관계없이 환자로 관리하게 된다. 본연구에서는 한국인 185명의 감염자와 140명의 비감염자에 대하여 정기적으로 CD4 및 CD8T 림프세포와 CD4/CD8비를 측정하였다. 시험은 Flow cytometer(Facstar)를 이용하여 각각의 CD 분자에 대한 모노크로날 항체를 이용하여 2중혈광색소염색방법으로 측정하였다. HIV감염자의 CD4-T림프세로 절대수 및 백분율은 각각 462 및 18.2%이었는 반면, CD8의 수치는 1,170 및 47.0%이었다. 또한 CD4/CD8비는 0.43이었다. 이와는 대조적으로 비감염자의 경우, 한국인의 CD4의 평균세포수는 886, 백분율은 32.9%이었으며, CD8 세포수는 730, 백분율은 26.8 그리고 CD4/CD8비는 1.31이었다. 외국인과 한국인과의 면역지표수치를 비교하였을 때에 CD4세포수와 백분율, CD8의 백분율에서는 현저한 차이가 없었으나 외국인 비감염자의 경우 CD4백분율이 43.6%, CD8 T림프세포의 절대수가 560으로 한국인과 약간의 차이가 있었다. 따라서 HIV감염자관리를 위한 면역지표측정시험에서의 각종수치의 정확한 해석을 위하여서는 한국인 비감염자수치를 고려해야할 것으로 판단된다. Several studies showed that the immunological factors such as CD4+ cell number, CD4%, CD8+ cell number and CD4/CD8 ratio and the serological factors such as β²-microglobulin(β²-MG), neopterin, soluble CD4, and soluble CD8 are related to the risk of development of AIDS. Especially, the CD4+ cell counts have been used to monitor progresson of HIV disease, to stratify, and to follow patients in clinical trials. Recently, the Centers for Disease Control and Prevention(CDCP) in USA has made the CD4+ cell count as a part of the classification of HIV disease. It is composed of 3 categories such as 1, 2, and 3 which asr ≥500/㎣, 200/㎣ ≥and <500/㎣, and < 200/㎣, respectively. In this study, to estimate the differences of immunological factors between HIV-infected and normal human groups in Korea, CD4+ T and CD8+ T cells, and the CD4/CD8 ratio were measured in 185 HIV-infected subjects and 140 healthy adult subjects. The lymphocyte subsets such as CD4+ T and CD8+ T were analysed by flow cytometer(FACStar) with two-color immunofluorescent stain using monoclonal antibodies such as anti-CD4 and anti-CD8 antibodies. The absolute numbers and percentages of CD4+ T and CD8+ T and the CD4/CD8 ratio of HIV infected persons were 462±277/㎣, 18.2±7.7%, 1,170±534/㎣, 47.0±10.6% and 0.43±0.26 whereas those of uninfected persons were 886±299/㎣, 32.9±7.0%, 730± 259/㎣, 26.8±6.4% and l.31±0.46(P<0.01). In addition, estimating the reference values of peripheral blood lymphocyte subsets of Korean, the absolute numbers and percentages of CD4+ T and CD8+ T and the CD4/CD8 ratio of 140 healthy adults persons were measured and compared with those of foreigners. The reference ranges of CD4+ T cells, CD8+ T cells, CD4%, CD8%, and the CD4/CD8 ratio and 1.31t0.46, respectively. The significant differences were not observed when compared with those of foreigners. However a little difference was observed in the percentages of CD4+ T and the absolute numbers of CD8+ T between the normal values of Korean and those of foreigners were 43.6±8.9%, 560±230/㎣. This result can also be useful as a basic data for the treatment and surveillance of HIV-infected patients in Korea.