대장균에서 인간의 면역 인터페론을 tac 프로모터를 이용하여 고도로 발현시키는 연구

곽규범,현형환,권병세,김광수 ( Kyu Bum Kwack,Hyung Hwan Hyun,Byung S . Kwon,Kwang Soo Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.3

The cDNA for the structural sequence of mature human mmune-interferon (IFN-γ) was introduced into E. coli high expression vector, pKK223-3,constructed by using tac promoter and rrnBT1T2 transcription terminator region and transformed to host, E. coli JM105, which has lacIq repressor gene on chromosomal DNA, E. coli MM294, and E. coli VMC9, which have laI repressor gene. The resultant ampicillin resistant transformants were analyzed by the immunoblotting and electron microscope. IFN-γ, which cross-reacted with mouse anti human IFN-γ serum, was produced as the soluble and the insoluble inclusion form. Their productions were efficiently regulated in E. coli JM105, but not in E. coli MM294, and E. coli YMC9 by using the IPTG.

High-level Expression of Human Immune Interferon in Escherichia coli by using tac Promoter

곽규범,현형환,권병세,김광수,Kwack, Kyu-Bum,Hyun, Hyung-Hwan,Kwon, Byung-S.,Kim, Kwang-Soo 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.3

인간의 면역 인터페론을 코딩하는 유전자를 tac 프로모터와 rrnBT1T2 전사중지서열을 갖는 프라스미드 pKK223-3에 클론닝하여 체세포 유전자에 lacIq 제한차서열을 갖는 대장균 JM105와 체세포 유전자에 lacIq 제한자서열을 갖는 대장균 MM294와 대장균 YMC9에 각각 형질전환시켰다. 생성된 형질전환주를 immunoblotting과 생물학적 역가분석, 전기영동 그리고 전자현미경을 사용하여 확인했다. 면역 인터페론의 발현은 대장균 JM105에서 효율적으로 조절되었으며, 대장균 MM294와 대장균 YMC9에서는 IPTG의 처리없이도 생산됨을 발견하였다. 또한 면역 인터페론이 용해된 형태와 불용성 상태인 inclusion body로 생성됨을 알았다. 이때 생산성은 $9.7{\times}10^9IU/l$이며 면역 인터페론 단백질이 대장균의 전체 단백질중 약 30%에 해당함을 알았다. The cDNA for the structural sequence of mature human mmune-interferon ($IFN-{\gamma}$) was introduced into E. coli high expression vector, pKK223-3, constructed by using tac promoter and rrnBT1T2 transcription terminator region and transformed to host, E. coli JM105, which has lacIq repressor gene on chromosomal DNA, E. coli MM294, and E. coli VMC9, which have laI repressor gene. The resultant ampicillin resistant transformants were analyzed by the immunoblotting and electron microscope. $IFN-{\gamma}$, which cross-reacted with mouse anti human $IFN-{\gamma}$ serum, was produced as the soluble and the insoluble inclusion form. Their productions were efficiently regulated in E. coli JM105, but not in E. coii MM294, and E. coli YMC9 by using the IPTG.

임신성 당뇨환자에서 실시간 중합효소연쇄반응을 이용한 미토콘드리아 DNA copy수의 정량적 분석과 미토콘드리아 A3243G 돌연변이 분석

장성운 ( Sung Woon Chang ),이숙환 ( Sook Hwan Lee ),전혜선 ( Hye Sun Jun ),곽규범 ( Kyu Bum Kwack ),조성원 ( Sung Won Cho ),정혜진 ( Hye Jin Jeong ),안소연 ( So Yeon Ahn ),김유리 ( You Lee Kim ),김수희 ( Soo Hee Kim ),박이석 ( Lee 대한산부인과학회 2005 Obstetrics & Gynecology Science Vol.48 No.4

사람 중간엽줄기세포 성장에 미치는 basic fibroblast growth factor의 영향

김성수(Sung Soo Kim),최정원(Jung Won Choi),곽규범(Kyu Bum Kwack),이영돈(Young Don Lee),서해영(Haeyoung Suh-Kim) 대한해부학회 2004 Anatomy & Cell Biology Vol.37 No.6

중간엽줄기세포는 중배엽유래의 세포로 분화할 수 있는 다분화능을 갖는 줄기세포로서 골수에서 쉽게 채취할 수 있다. 본 연구에서는 사람의 중간엽줄기세포를 시험관내에서 장기간 배양시 세포의 증식과 다분화능에 있어서 basic fibroblast growth factor (bFGF)의 영향을 조사하였다. 세포를 배양할 수 있는 최적의 조건을 설정하기 위하여 여러 성장인자를 시험하였다. Hepatic growth factor (HGF)와 leukemia inhibitory factor (LIF)는 골수에서 분리한 중간엽줄기세포의 증식에 별다른 영향을 주지 않았으나, bFGF는 시험관내에서 세포의 성장을 3배 정도 증가시켰으며, 그 효과에 있어서 bFGF의 농도가 10 ng/mL 이상인 경우에는 큰 차이가 없었다. 저연령층의 골수에서 추출한 중간엽줄기세포는 bFGF의 존재와 상관없이 지방세포, 뼈세포, 연골세포로 분화하는 잠재력을 유지하고 있었으며 CD9, CD13, CD15, CD90, CD137, CD140b에 양성이었고, CD14, CD34, CD45에는 음성이었으며, 정상적인 핵형을 유지하고 있었다. 반면에 고령의 공여자로부터 분리한 중간엽줄기세포는 bFGF가 없을 경우 지방세포로의 분화능이 현격히 낮았으나, bFGF가 있는 경우에는 지방세포 및 뼈세포로의 분화능이 유지되었다. 또한 bFGF가 첨가된 배양액에서 증식시킨 중간엽줄기세포는 미분화상태에서도 이미 신경줄기세포의 표지자인 nestin과 신경세포의 표지자인 β-tubulin III을 발현하고 있었다. 이러한 결과는 bFGF가 사람의 중간엽줄기세포의 증식과 다분화능을 유지시키는데 중요한 성장인자로 작용함을 의미하고 있다. 또한, 중간엽줄기세포를 신경계질환의 세포치료제로서 사용하기 위해서는 미분화상태와 신경세포로 분화된 후를 구별할 수 있는 새로운 표식인자가 필요함을 제시하고 있다. Human mesenchymal stem cells (hMSCs) are multipotent stem cells that can differentiate into several mesenchymal lineage cells. In this study, we established conditions that allowed a long term expansion of hMSCs. To search for the optimum culture condition, growth rates of hMSCs were measured in the presence of several growth factors. Hepatic growth factor (HGF) and leukemia inhibitory factor (LIF) did not facilitate proliferation of hMSCs. In contrast, basic fibroblast growth factor (bFGF) effectively promoted growth of the cells in vitro by 3 fold. The growth stimulatory effect of bFGF was dependent on the concentration. The adipogenic potential was dramatically decreased in hMSCs isolated from an aged donor whereas osteogenic potential was minimally decreased. Addition of bFGF resumed the adipogenic and osteogenic differentiation potential. Thus, the cells that expanded in the presence of bFGF retained the potential to differentiate into adipogenic, chondrogenic, or osteogenic lineage cells. MSCs could be expanded for at least 8 passages with bFGF and the resulting cells retained the normal karyotype. The cells were positive for CD9, CD13, CD15, CD90, CD137, and CD140b; but negative for CD14, CD34, and CD45. Importantly, the cells were found to express a neural stem cell marker, nestin, and a neuronal marker, β-tubulin III. The results suggest that bFGF promote proliferation while maintaining multi-lineage differentiation potency of hMSCs. Finally, we suggest that it is critical to identify novel markers other than nestin or β-tubulin III to monitor acquisition of neuronal phenotypes by hMSCs.

한국인 위암 발병과 H-RAS 유전자 다형성의 연관성 연구

송희진 ( Hee Jin Song ),편정아 ( Jung A Pyun ),이광재 ( Kwang Jae Lee ),조성원 ( Sung Won Cho ),곽규범 ( Kyu Bum Kwack ) 대한소화기학회 2010 대한소화기학회지 Vol.56 No.2

Background/Aims: Oncogenic RAS gene mutations have been frequently observed in many tumor types, and their associations with various cancers were reported. This study was conducted to evaluate the association between H-RAS T81C polymorphism and gastric cancer development. Methods: H-RAS T81C polymorphism was genotyped in 321 chronic gastritis (ChG) and 151 gastric cancer (GC) patients using GoldenGate(R) Assay kit. Logistic regression analysis adjusted for age and gender was performed to identify the differences of genotype and allele distributions between the each group. Results: All ChG and GC patients were in Hardy-Weinberg equilibrium. When the frequencies of H-RAS T81C genotype in each group were compared, the homozygous type of major allele TT was more frequent in GC group (62.9%) than ChG group (57.3%), while the frequencies of heterozygous type TC and homozygous type of minor allele CC were higher in ChG group than GC group (39.3% vs. 33.8%, 3.4% vs. 3.3%, respectively). In the results of logistic regression analyses adjusted for age and gender, the odds ratios were 0.845 (0.604-1.182), 0.799 (0.556-1.147), 0.741 (0.493-1.114) and 1.094 (0.366-3.270) for allele, codominant, dominant and recessive models, respectively. However, significant difference was not observed between two groups in any models. Conclusions: H-RAS T81C polymorphism was not associated with gastric cancer development in a Korean population. (Korean J Gastroenterol 2010;56:78-82)

위장관 ; MMP7, MMP8, MMP9 유전자의 단일염기다형성과 위암 발생 및 림프절 전이와의 연관성 연구

김지혜 ( Ji Hye Kim ),편정아 ( Jung A Pyun ),이광재 ( Kwang Jae Lee ),조성원 ( Sung Won Cho ),곽규범 ( Kyu Bum Kwack ) 대한소화기학회 2011 대한소화기학회지 Vol.58 No.5

Background/Aims: Matrix metallopeptidase (MMP) is known to be involved in tumor invasion and metastasis of cancer. This study investigated the association of MMP7 rs11568818, MMP8 rs11225395, MMP9 rs17576 and rs2250889 with gastric cancer (GC) development and lymph node metastasis (LNM). Methods: Samples were obtained from 326 chronic gastritis (CG) and 153 GC patients and genotyped by using the GoldenGate(R) method. Chi-square test was performed to identify the difference of allele distribution between each group (CG vs. GC; CG vs. with LNM GC). The associations of genotype with risk of GC and LNM were estimated by odds ratio and the 95% confidence interval was calculated by logistic regression adjusting for age and sex. Results: The allele and genotype frequencies of MMP7 rs11568818, MMP8 rs11225395, MMP9 rs17576 and rs2250889 were not associated with the development of GC and LNM. Conclusions: In summary, MMP7 rs11568818, MMP8 rs11225395 MMP9 rs17576 and rs2250889 were not associated with the GC development and LNM in Korean population. (Korean J Gastroenterol 2011;58:245-251)

한국인 자폐증과 Chromosome 5p14에 존재하는 CDH9, CDH10 유전자 다형성의 연관성 연구

이애리(Aeri Lee),박정원(Jung Won Park),남민(Min Nam),방희정(Hee Jung Bang),양재원(Jae Won Yang),최경식(Kyung-Sik Choi),김수강(Su Kang Kim),정주호(Joo-Ho Chung),곽규범(Kyu Bum Kwack) 대한소아청소년정신의학회 2011 소아청소년정신의학 Vol.22 No.4

Objectives:The region of chromosome 5p14 is known to be associated with autism spectrum disorder (ASD). The cadherin9 (CDH9) and cadherin10 (CDH10) genes are located in the region of chromosome 5p14 and reported to be associated with ASD in the Caucasian population. We performed an association study to identify if single nucleotide polymorphisms (SNPs) located on the CDH9 and CDH10 genes are associated in the Korean population. Methods:Genomic DNA was extracted from the blood of 214 patients with ASD and 258 controls. SNPs selected from two genes were genotyped using an Illumina Golden-Gate Genotyping assay with VeraCode technology. Statistical analysis was performed using SAS and Plink software. Results:All controls and ASD patients were in Hardy-Weinberg equilibrium. In the results of logistic regression analyses for the genotype model and the chi-square test for the allele model, we found that SNPs on the CDH9 and CDH10 genes were not associated with ASD. Conclusion:Our data suggests that the CDH9 and CDH10 genes are not associated with ASD in the Korean population.

유전자 조작 알파 인터페론의 조직분포에 관한 연구

김제학,이혜선,김달현,조남진,곽규범 한국약제학회 1987 Journal of Pharmaceutical Investigation Vol.17 No.4

The distribution features of recombinant human alpha-interferon (rHuIFN- αA) and ^(14)C-radiolabeled rHuIFN- αA (^(14)C-rHuIFN- αA) were investigated in ICR mice after i.v. injection. The level of rHuIFN- αA in the kidney was significantly higher than those in lung and liver at 10min after the injection. But the level was reduced significantly at 60min. The level of radioactivity in the kidney was also significantly higher than those in other organs after i.v. injection of ^(14)C-rHuIFN- αA, but it was reduced at much slower speed than was rHuIFN- αA. These results show that interferon is distributed repidly and the kidney is the main site of distribution and metabolism of rHuIFM- αA.