RFLP Analysis of cag7 Gene of Helicobacter pylori

강형련,최미영,김경미,김두수,관영철,박승규,황향란,송재영,백승철,이욱곤,윤희상,재명,박정욱,이광호 대한미생물학회 2004 Journal of Bacteriology and Virology Vol.34 No.3

.The cag7 gene of Korean H. pylori strains was analyzed by RFLP to develop a discriminatory tool for genotyping clinical isolates. For this study, a total of 82 H. pylori strains were isolated from the patients; 27 strains from the patients with chronic gastritis, 26 from duodenal ulcer, and 29 from gastric cancer. Genomic DNA was isolated and subjected to PCR targeting entire ORF or the repeat regions I and II of cag7 gene. PCR products from entire ORF or repeat region I of cag7 gene were divided into two types. However, there was no difference in the length of PCR products from the repeat region II. By the PCR genotyping of the entire cag7 gene, genotypes A and B were established, which showed approximately 5,100 and 5,500 bp PCR products, respectively. The repeat region I showed approximately 600 or 1,000 bp DNA fragments by PCR. The length of cag7 gene was determined by the size variation in the repeat region I. In addition, RFLP analysis of the PCR products of cag7 gene showed 11 subtypes, based on the major bands. These findings illustrate that the genetic diversity of the repeat region I would serve a reliable target for the genotyping of the cag7 gene.

An Easy Way for the Rapid Purification of Recombinant Proteins from Helicobacter pylori Using a Newly Designed Expression Vector

강형련,조진성,권순욱,송재영,서지현,조명제,백승철,윤희상,이광호,이우곤 한국미생물학회 2014 The journal of microbiology Vol.52 No.7

We constructed a H. pylori expression vector which consisted of both a His-tag and a GST tag as purification toolsfor recombinant protein and a chloramphenicol resistant catgene as a reporter. The backbone of the vector pBK contained an ColEI origin of replication and a kanamycin resistantgene. A set of oligos for the His-tag and the PCR product of gst (glutathione S-transferase) gene were inserted sequentiallyin frame in themulti-cloning site of pBK. The orf of cat was inserted downstream of the gst to generate pBKHGC. The 3' part of H. pylori clpB and flaA were cloned into the vector which was introduced into H. pylori. Recombinant proteins were purified by GSH affinity column, digested with thrombin and were analyzed by western blotting. The final recombinant proteins were successfully purified.

Proteomic Analysis of Helicobacter pylori Whole Cell Proteinsusing the Narrow Range IPG Strips

박정원,이성규,송재영,전진수,주정수,윤희상,서지현,강형련,백승철,이우곤,조명제,이광호 대한미생물학회 2007 Journal of Bacteriology and Virology Vol.37 No.4

It has been reported that most of Helicobacter pylori proteome components appear so crowded in the region of pH 4.5~8.0 that a lot of them were inseparable in 2-DE using the broad range IPG strip. Therefore, inseparable protein spots in 2-DE profiles have to be apart from each other for improving the protein identification. Here, we attempt to examine the usability of the narrow range IPG strips for separating close spots in the broad range IPG strip at proteomic analysis of H. pylori. The whole cell proteins of H. pylori strain 26695 were separated by narrow range IPG strips (pI 3.9~5.1, 4.7~5.9, 5.5~6.7, and 6.3~8.3, respectively), followed by SDS-PAGE, and visualized by silver staining, showing that the distances between spots were widened and the total number of detectable spots was increased. Resolved protein spots were identified by the peptide fingerprinting using MALDI-TOF-MS. As a result, 87 expressed proteins were identified by the peptide fingerprinting. Of them, 23 proteins, including hydrogenase expression/formation protein, purine-binding chemotaxis protein, and ribosomal protein S6, have not been reported in the previous proteome studies of H. pylori. Thus, these results demonstrate that the high complexity proteome components could be effectively separated using the narrow range IPG strips, which might be helpful to strengthen the contents of the master protein map of the H. pylori reference strain.

Anti-Helicobacter pylori Compounds from Maackia amurensis

박우성,Ji-Yeong Bae,김민갑,이우곤,강형련,백승철,Kyung Mook Lim,이미경,안미정,김혜진 한국생약학회 2015 Natural Product Sciences Vol.21 No.1

Eight isoflavonoid compounds were isolated from the EtOAc fraction of Maackia amurensis which had shown the highest anti-Helicobacter pylori activity among the fractions, using medium pressure liquid chromatography and recrystallization. Based on the spectroscopic data including 1H-NMR, 13C-NMR, HMBC and MS data, the chemical structures of the isolates were determined to be (-)-medicarpin (1), afromosin (2), formononetin (3), tectorigenin (4), prunetin (5), wistin (6), tectoridin (7) and ononin (8). Anti-H. pylori activity of each compound was evaluated with broth dilution assay. As a result, (-)-medicarpin (1), tectorigenin (4) and wistin (6) showed anti-H. pylori activity. (-)-Medicarpin (1) exhibited the most potent growth inhibitory activity against H. pylori with the minimal inhibitory concentration (MIC)90 of 25 mM, and tectorigenin (4) with MIC90 of 100 mM ranked the second. This is the first study to show the anti-H. pylori activity of M. amurensis, and it is suggested that the stem bark of M. amurensis or the EtOAc fraction or the isolated compounds can be a new natural source for the treatment of H. pylori infection.

Proteomic Analysis of Thiol-active Proteins of Helicobacter pylori 26695

박정원,송재영,황향란,박희진,윤희상,강형련,이곤호,백승철,이우곤,조명제,이광호,서지현 대한미생물학회 2012 Journal of Bacteriology and Virology Vol.42 No.3

Helicobacter pylori are a capnophilic bacterium, which colonize gastric mucosa and are resistant to acidic and oxidative damage. Thiol-active proteins subserve redox functions in tolerating oxidative stress and environmental toxicants, such as hydrogen peroxide and hypochlorous acid. We analyzed disulfide-containing proteins of H. pylori strain 26695. Active disulfide-containing proteins were separated by thiol-affinity chromatography, displayed with two-dimensional electrophoresis (2-DE), and identified by MALDI-TOF-MS. Thirty-five putative disulfide proteins, including AhpC (HP1563), GroEL (HP0011), and FrdB (HP0191), were identified in this study. In addition, 4 disulfide proteins of HypB, FusA, TufB, and AhpC showed enhanced intensities in the periplasmic space when compared with the pellet,suggesting that these proteins might play roles in the first redox system against environmental oxidative stresses. Disulfide-containing proteins identified in this study will provide the standard landscape for constructing the proteome components responsible for redox regulation of H. pylori.

Comparison of the Antibiotic Resistance of Helicobacter pylori Isolated in Jinju Over a 15-year Period

서지현,구상일,윤희상,전진수,임재영,박찬후,우향옥,강형련,백승철,이우곤,조명제,이광호 대한미생물학회 2012 Journal of Bacteriology and Virology Vol.42 No.4

The aims of this study were to investigate the changing pattern of Helicobacter pylori antibiotic resistance in Jinju over a 15-year period. H. pylori strains were isolated from 170 adults living in Jinju from 1985-1989, 1990-1994 and 1995-1999, and from 23 adults living in Cheongju from 1995 to 1999. Susceptibility to erythromycin, clarithromycin,azithromycin, amoxicillin, tetracycline, metronidazole, furazolidone, levofloxacin, ciprofloxacin, moxifloxacin, and rifabutin was tested using the serial two-fold agar dilution method. Moxifloxacin resistance significantly increased in Jinju from 1985-1989 (0%) to 1995-1999 (14.9%) (p < 0.0001). Resistance to amoxicillin was increasesed trend to decreased trend from 1985 to 1999 (p = 0.033), whereas metronidazole resistance decreased from 37.5% to 21.3%. Resistance to furazolidone was greater from 1985-1989 (9.4%) than in 1995-1999 (2.1%). In comparing Jinju and Cheongju, minimal inhibitory concentrations (MICs) of tetracycline and levofloxacin among H. pylori isolated from Jinju were lower than for isolates from Cheonju (p < 0.05). The levofloxacin resistance rate was higher in Cheongju than in Jinju (p = 0.02). No macrolide resistance was observed in Cheongju. Overall, we did not observe any remarkable antimicrobial resistance increase of H. pylori strains isolated from Jinju over 15 years. The MIC distributions of antimicrobials and antimicrobial resistant rates were time- and region-specific among different strains. Future anti-H. pylori eradication regimens should be designed based on the changing patterns of antimicrobial resistance according to the resident area.

Profiling of the Bacteria Responsible for Pyogenic Liver Abscess by 16S rRNA Gene Pyrosequencing

송윤규,심상군,김광민,이동해,김대수,최상행,송재영,강형련,백승철,이우곤,조명제,이광호 한국미생물학회 2014 The journal of microbiology Vol.52 No.6

Pyogenic liver abscess (PLA) is a severe disease with considerablemortality and is often polymicrobial. Understandingthe pathogens that cause PLA is the basis for PLA treatment. Here, we profiled the bacterial composition in PLA fluid bypyrosequencing the 16S ribosomal RNA (rRNA) gene basedon next-generation sequencing (NGS) technology to identifyetiological agents of PLA and to provide information oftheir 16S rRNA sequences for application to DNA-basedtechniques in the hospital. Twenty patients with PLA whounderwent percutaneous catheter drainage, abscess culture,and blood culture for isolates were included. Genomic DNAsfrom abscess fluids were subjected to polymerase chain reactionand pyrosequencing of the 16S rRNA gene with a454 GS Junior System. The abscess and blood cultures werepositive in nine (45%) and four (20%) patients, respectively. Pyrosequencing of 16S rRNA gene showed that 90% of thePLA fluid samples contained single or multiple genera ofknown bacteria such as Klebsiella, Fusobacterium, Streptococcus,Bacteroides, Prevotella, Peptostreptococcus, unassignedEnterobacteriaceae, and Dialister. Klebsiella was predominantlyfound in the PLA fluid samples. All samples thatcarried unassigned bacteria had 26.8% reads on average. We demonstrated that the occurrence of PLA was associatedwith eight known bacterial genera as well as unassignedbacteria and that 16S rRNA gene sequencing was more usefulthan conventional culture methods for accurate identificationof bacterial pathogens from PLA.

상부 위장관 증세가 있는 소아의 위십이지장병변 및 Helicobacter pylori 감염

윤영란,김미령,임재영,최명범,박찬후,우향옥,윤희상,고경혁,강형련,백승철,이우곤,조명제,이광호,Yoon, Young-Ran,Kim, Mi-Ryeung,Lim, Jae-Young,Choi, Myoung-Bum,Park, Chan-Hoo,Woo, Hyang-Ok,Youn, Hee-Shang,Ko, Gyung-Hyuck,Kang, Hyung-Lyun,Baik, S 대한소아소화기영양학회 2003 Pediatric gastroenterology, hepatology & nutrition Vol.6 No.2

목 적: 만성 반복성 복통, 식사 후 상복부 불쾌감, 잦은 구토나 구역질이 있는 소아를 대상으로 위내시경을 시행하여 위십이지장 병변을 확인하고, 생검체를 이용한 위십이지장 조직학적 검사와 H. pylori 검출 그리고 면역블롯팅을 통해 혈청 내에 H. pylori 특이 항체 존재를 확인하여 한국에서 관찰되는 소아의 위장관 증세와 위십이지장병변 및 H. pylori 감염과의 관계를 조사하고자 하였다. 방 법: 1990년 6월부터 1991년 4월까지 경상대학교병원 소아과에서 상부 위장관 증상으로 위내시경을 시행 받은 184명 중 위 전정부에서 생검이되었고, 요소분해효소 검사, Warthin-Starry 은염색 혹은 Hematoxylin-Eosin 염색으로 조직학적에서 H. pylori의 존재유무를 확인할 수 있었던 107명을 대상으로 위십이지장 조직학적 검사와 IgG 면역블롯팅에 의한 항-H. pylori 항체 보유 유무를 확인하였다. 결 과: 1) 대상 환아 107명 중 남아가 61명(57%), 여아가 46명(43%)이었으며, 연령은 2세부터 15세까지 분포하였고 평균연령은 10.7세로서 10세에서 15세 사이가 가장 많았다. 2) 내시경상 15%에서 위출혈 반점, 위궤양, 십이지장궤양, 십이지장 미란, 출혈성 십이지장염 등이 관찰되었고 대부분은 다양한 정도의 위점막 발적이 관찰되었다. 3) 107명 중 94명(88%)에서 경도 이상의 조직학적 만성위염이 있었으며, 십이지장 조직이 검사 가능하였던 99명 전원에서 만성십이지장염이 있었다. 4) 요소분해효소 검사는 위에서는 45%, 십이지장에서는 25.6%에서 양성으로 판정되었다. H&E 염색 검사에서 38.7%, Warthin-Starry 은염색 검사에서는 40%에서 HPLO 양성이었다. 이 세가지 검사 중 1개 검사에서 양성인 경우인 조직학적 H. pylori 양성은 57%이었다. 5) IgG 면역블롯팅 양성은 96%이었다. 6) 연령군별 조직학적 H. pylori 양성은 0∼4세 군에서는 29%, 5∼9세 군에서는 41%, 10∼15세 군에서는 68%로 연령이 증가할수록 양성률이 증가하였으나, 조직학적 만성위염 및 만성십이지장염 빈도와 면역블롯 양성 빈도는 연령군별 차이가 없이 높은 양성률을 유지하였다. 결 론: 상부 위장관 증세가 있는 소아의 대부분은 조직학적 만성위염 및 만성십이지장염과 동시에 H. pylori에 대한 특이 IgG 항체를 보유하고 있었다. Purpose: This study was undertaken to evaluate the gastroduodenal pathology and Helicobacter pylori infection in children with upper gastrointestinal symptoms. Methods: One hundred and seven pediatric patients with upper gastrointestinal symptoms were undergone endoscopy at the Gyeongsang National University Hospital from June 1990 to April 1991. Histopathologic examination was done by H & E staining of gastric antral biopsy specimen and gastritis was defined according to the Sydney System. Tissue H. pylori status was evaluated with the urease test using Christensen's urea broth and H & E or Warthin-Starry silver staining of gastric antral biopsy specimen. IgG Immunoblotting were also performed to detect specific anti-H. pylori antibody in these patients. Results: The reasons for endoscopy were recurrent abdominal pain, acute abdominal pain, sallow face, hunger pain, and frequent nausea. Variable degrees of gastric mucosal hyperemia were found in most of the patients. Gastric hemorrhagic spots, gastric ulcer, duodenal ulcer, duodenal erosion, and hemorrhagic duodenitis were rare endoscopic findings. Histologic chronic gastritis was found in 88% of 107 patients. Histologic chronic duodenitis was observed in all 99 patients whose tissue were available. Gastric tissue H. pylori was positive in 57% of 107 patients by one of the ureasetest, H & E staining and Warthin-Starry silver staining. However, gastric tissue H. pylori detection rate was lower in the younger age groups. Anti-H. pylori IgG antibodies were detectable in 96% of 107 patients. Conclusion: Chronic gastroduodenitis and anti-H. pylori IgG antibody were ubiquitous in children with upper gastrointestinal symptoms.

보존제 없이 장기간 냉동 보관된 생검 조직에서의 Helicobacter pylori 배양

윤희상,구민지,전진수,서지현,박은실,임재영,박찬후,우향옥,고경혁,강형련,백승철,이우곤,조명제,이광호 대한소화기학회 2006 대한소화기학회 추계학술대회 Vol.2006 No.-

대장균에서 발현된 A군 로타바이러스 VP6 단백질을 이용한 로타바이러스 감염의 혈청학적 진단의 유용성

서지현,김소영,박지숙,임재영,박찬후,우향옥,윤희상,김원용,강형련,백승철,이우곤,조명제,이광호,Seo, Ji-Hyun,Kim, So-Young,Park, Ji-Sook,Lim, Jae-Young,Park, Chan-Hoo,Woo, Hyang-Ok,Youn, Hee-Shang,Kim, Won-Yong,Kang, Hyung-Lyun,Baik, Seung-Chul 대한소아소화기영양학회 2010 Pediatric gastroenterology, hepatology & nutrition Vol.13 No.2

목 적: 로타바이러스 감염역학의 변화를 연구하기 위해 A군 로타바이러스의 VP6 유전자를 대장균에 발현시켜 확보한 rVP6 단백질이 항원성이 있는지를 확인하고 이것을 항원으로 한 효소면역측정법이 로타바이러스 IgG, IgA와 IgM 항체를 정량적으로 평가할 수 있는지 확인하고자 하였다. 방 법: 경상대학교병원에서 로타바이러스 감염을 진단받은 소아들 중 진단 받기 전, 진단 당시, 회복기 이후의 연속적인 혈청을 확보할 수 있었던 22명에게서 100개의 혈청을 경상대학교병원 인체자원은행으로부터 제공받아 로타바이러스 VP6 유전자를 클로닝 하여 대장균에 발현시켜 제조한 rVP6 항원으로 한 효소면역측정법으로 IgG, IgA와 IgM 항체 역가를 측정하였다. 이 중 건강한 신생아 4명에서는 면역 블로팅을 같이 시행하였다. 결 과: 건강한 신생아와 영유아 17명에서 감염 후 확보된 혈청에서 IgG, IgA, IgM 항체 중 최소한 한 종류의 항체 역가 증가가 동반되어 있었다. 면역이 저하된 소아 5명 중 4명에서는 IgG 항체 역가는 증가되었으나 IgA 항체 역가는 2명에서만 증가하였고, IgM 항체 역가는 5명 모두 증가하지 않았다. 신생아 4명에서 시행된 면역 블로팅 검사에서는 IgM 항체인 경우는 효소면역측정법보다 예민하게 진단 초기부터 4명 모두 양성으로 판정되었다. 결 론: A군 로타바이러스의 VP6 유전자를 대장균에 발현시켜 확보한 rVP6 단백질은 항원성이 있으며 이것을 항원으로 한 효소면역측정법은 로타바이러스 감염후 IgG, IgA, IgM 항체 역가 증가를 정량적으로 평가할 수 있어 지역사회에서 발생한 로타바이러스 감염역학의 변화를 연구하는데 유용할 것으로 판단된다. Purpose: The serologic diagnosis of rotaviral infections is not commonly used in clinical practice, but is used in seroepidemiologic studies. In this study, the usefulness of Escherichia coli-expressed recombinant VP6 proteins of group A rotavirus in the serodiagnosis of rotavirus infections by ELISA was evaluated. Methods: The recombinant VP6 proteins of group A rotavirus expressed in E. coli Rosetta II strain were purified and identified. One hundred sera from 22 children (4 healthy neonates, 13 healthy children, and 5 immunocompromised children) who had serial sera samples prior to and after rotavirus infections were provided by the Gyeongsang National University Hospital, a member of the National Biobank of Korea. IgG, IgA, and IgM antibodies against rVP6 were analyzed by ELISA in all of the patients and Western blot analysis in 4 neonates. Results: ELISA tests using rVP6 proteins of group A rotavirus as antigen revealed that IgG, IgA, and IgM antibodies increased after rotaviral infections in most neonates and healthy children. IgG antibodies also increased after rotaviral infections in most immunocompromised children without an adequate increase in IgM or IgA antibodies. Western blot analysis in four neonates revealed very early IgM antibody responses, even in the sera with low optical densities in ELISA tests. Conclusion: Our study showed that ELISA using rVP6 as an antigen is a valid diagnostic tool for seroepidemiologic studies of rotavirus infections and Western blot analysis is a sensitive test in detecting IgG, IgA, and and IgM antibodies in patients with rotavirus infections.