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안상훈,천지용,신수경,박준용,유왕돈,홍선표,김수옥,한광협 대한간학회 2013 Clinical and Molecular Hepatology(대한간학회지) Vol.19 No.4
Background/Aims: Molecular diagnostic methods have enabled the rapid diagnosis of drug-resistant mutations in hepatitis B virus (HBV) and have reduced both unnecessary therapeutic interventions and medical costs. In this study we evaluated the analytical and clinical performances of the HepB Typer-Entecavir kit (GeneMatrix, Korea) in detecting entecavir-resistance-associated mutations. Methods: The HepB Typer-Entecavir kit was evaluated for its limit of detection, interference, cross-reactivity, and precision using HBV reference standards made by diluting high-titer viral stocks in HBV-negative human serum. The performance of the HepB Typer-Entecavir kit for detecting mutations related to entecavir resistance was compared with direct sequencing for 396 clinical samples from 108 patients. Results: Using the reference standards, the detection limit of the HepB Typer-Entecavir kit was found to be as low as 500 copies/mL. No cross-reactivity was observed, and elevated levels of various interfering substances did not adversely affect its analytical performance. The precision test conducted by repetitive analysis of 2,400 replicates with reference standards at various concentrations showed 99.9% agreement (2398/2400). The overall concordance rate between the HepB Typer-Entecavir kit and direct sequencing assays in 396 clinical samples was 99.5%. Conclusions: The HepB Typer-Entecavir kit showed high reliability and precision, and comparable sensitivity and specificity for detecting mutant virus populations in reference and clinical samples in comparison with direct sequencing. Therefore, this assay would be clinically useful in the diagnosis of entecavir-resistance-associated mutations in chronic hepatitis B. (Clin Mol Hepatol 2013;19:399-408)
전립선 암조직에서의 사람피필로마바이러스의 감염과 p53단백질의 발현에 대한 연구
권두환,진승원,강병태,윤희식,유왕돈,김현수,이상숙,이호자,박순희 대한바이러스학회 1996 Journal of Bacteriology and Virology Vol.26 No.2
Prostatic carcinoma is the leading second cause of cancer in men. Previous epidermiological studies implicated human papillomavirus as an infectious agent. Since there are only limited studies on the association of HPV to prosate cancer, we examined the prevalence of HPV infections in korean prostate cancer patients. We observed that out of 26 cases, 4 cases and 5 cases were infected by HPV 16(27%) and HPV 18 (31%), respectively and 3 cases by both (46%) and at least 18 were positive for HPV (69%). For these samples, immunohistochemical detection of the p53 and proliferative cell nuclear antigen (PCNA) were also studied, using monoclonal antibodies. Sixteen of 26 (61%) showed immunostaining for p53 protein. While 8 samples with no HPV infection (100%) showed all positive for p53 protein staining, less than half of the 18 patients with any HPV infection (44%) showed p53 protein staining. These findings indicate that altered expression of p53 protein occurs in the more than half of prostate cancers, however, p53 expression is less frequent in HPV infected tissues. This implies that there might be an inverse correlation in general between HPV infection and p53 amplification. However, while 50% (4 of 8) of HPV negative prostate cancer was positive for PCNA staining, 13 out of 18 HPV infected patients (72%) were positive. Therefore HPV infection is more strongly associated with increase proliferation. In addition HPV infected cancer patients are generally in more advanced status implying that HPV infection plays a role in the development of highly malignant prostatic car- cinomas, eventhough the statistical significance of this interpretation might be waited for the analysis of more cases.
베로 세포에서 생산된 2세대 일본뇌염 백신의 마우스에서의 면역원성
문상범,홍선표,김달현,김수옥,유왕돈,강재구,Kenneth H. Eckels 한국미생물학회 1999 미생물학회지 Vol.35 No.1
베로 세포에 적응시켜 개발된 약독화 일본뇌염 바이러스인 CJ5003 주를 이용해 제조된 새로운 2세대 일본뇌염 백신의 효과를 마우스에서 조사하였다. Adjuvant 로 aluminiu hydroxide gel을 사용한 경우, 그렇지 않은 경우에 비해 높은 일본뇌염 바이러스 특이 항체 유도능과 10배 향상된 중화항체 유도능을 보였으며, 항원의 양을 변화시켜 면역시킨 결과 0.5 ng 의 적은 항원양으로도 항체가 유발되었고 방어 가능한 수준인 1:10 이상의 중화항체가 유지되었다. 500ng의 항원양으로 면역된 마우스를 면역 초기부터 24주간 관찰한 결과 중화항체가가 14주까지 1:200 이상으로 계속 유지되었으며 24주에도 1:160을 유지하여 일본뇌염에 대한 방어효력이 장기간 유지되고 있음을 나타냈다. 또한 면역된 마우스 혈청은 Nakayama-NIH, SA14, P3 등 3종의 신경독성 일본뇌염 바이러스주에 대해서 각각 유사한 수준의 일본뇌염 바이러스 특이 항체가를 보여주어 다양한 일본뇌염 바이러스 주에 대한 방어 가능성을 보여주었다. 마우스를 이용한 뇌내 직접 공격 시험결과, CJ5003 후보 백신은 90% 이상의 높은 생존율을 나타냈다. 이상의 in vitro, in vivo 시험 결과는 베로 세포에서 생산된 CJ5003 후보 백신의 차세대 백신으로 개발 가능성을 시사한다. In this study, to evaluate newly developed Japanese encephalitis (JE) vaccine candidate CJ50003, we assessed its immunogenicity along with a previously commercialized inactivated JE Biken vaccine. The CR0003 viral antigens produced in Vero cells were administered suhcutaneouly to mice either with alum-adjuvanled or free form. The ELISA titers and neutralizing (NEUV antibody titers accounting for major protective immunity in JE were determined. Mice given alum-adjuvanted vaccine had a 10 times higher antigen-specific NEUT antibody response than did those which {lad received free antigens. This NEUT antibody response was maintained until day 168 with NEUT titer more than 1:160. Even with the 0.5 ng of alum-adjuvanted antigen dose, NEUT titer was induced more than 1:10 which is considered as an evidence for seroconversion and protection. Thc mice immune sera had a similar rate of cross-reactivity against three different viral antigens, Nakayama-NlH, P3 and SA14; as determined by ELISA assay. In a mice challenge model, vaccination with the GI50003 conferred more protection than with commercialized Biken vaccine against Nakayama virus. These data demonstrated that CJ50003 vaccine candidate has an excellent prophylactic efficacy and implicated it has a strong potential for further development and commercialization.
대장균에서 발현시킨 사람 파필로마 바이러스 18 형 E6 단백질의 분리정제와 사람 혈청내의 18 형 E6 항체 검색에의 이용
정태화,손우익,박순희,강주현,유왕돈,진승원 경북대학교 유전공학연구소 1995 遺傳工學硏究所報 Vol.10 No.1
Human papillomavirus type 16 and 18 encode two major transforming proteins E6 and E7. Recombinant E6 protein for human papillomavirus type 18(HPV 18) E6 gene was expressed in E. coli in large quantity as authentic size, non fused form of protein. However, the protein was produced in inclusion bodies. Therefore this bacterially produced E6 protein was solublized in SDS solution and purified to the homogeneity by gel filtration column chromatographies using Sephacryl S-200 column. The yield of purified 18 E6 recombinant protein was approximately 30 ㎎/ℓ liter culture. This purified recombinant 18E6 protein was used to determine the presence of antibodies that might have been produced against HPV 18 infections in the human sera. We tested and compared sera obtained from both cervical cancer patients and people having no apparent cervical cancers. These were accomplished using Western blot. Antibodies reactive to recombinant HPV 18E6 protein were frequently detected in cases with cervical carcinoma(14 out of 20, 70%), compared to people with no apparent carcinoma(4 out of 9, 44%).
곤충세포에서 사람파필로마바이러스 16형 L1 재조합 단백질의 생산 및 바이러스 유사 입자분리
강병태,신원,진승원,이희구,임종석,정현호,김현수,유왕돈,박순희 대한바이러스학회 1995 Journal of Bacteriology and Virology Vol.25 No.2
The L1 capsid protein of Human Papillomavirus(HPV) type 16 was expressed in Sf-21 insect cells via a baculovirus vector. The gene coding Ll ORF beginning at the second methionine was cloned into pBacPAK1 expression vector. Recombinant Ll protein was confirmed by 12% SDS- PAGE followed by Western blotting with monoclonal antibody against HPV 16L1. The L1 protein was expressed at high level and detected at the mobility of 56 Kda. But HPV 6bL1 antibody did not react with recombinant L1 protein. This indicates that the major capsid protein of HPV 16L1 is antigenically different to the HPV 6bL1. In addition, L1 major capsid proteins self-assembled into virus like particles(VLPs) and could be purified in preparative amounts by CsC1 density gradient, ultracentrifugation indicating that L1 protein alone is sufficient for virus like particle assembly in insect cells.
형질전환체 담배에서 사람 파필로마 바이러스 HPV-18 E6 유전자의 발현
김현수,김달웅,손우익,현미아,박순희,유왕돈,전재필 경북대학교 유전공학연구소 1995 遺傳工學硏究所報 Vol.10 No.1
To develop a HPV-18 oral vaccine, HPV-18 E6 gene was cloned into an expression vector, under the control of CaMV 35S dual promoter and 5'UT region of TEV. The constructed vector (pUS-E6) was used to transform tobacco plants by the Agrobacterium-mediated leaf disc method. The transformants were then regenerated to whole transgenic plants. The HPV-18 E6 gene was found to be expressed in the transgenic tobacco plants as determined by PCR, RT-PCR, Northern analysis and Western analysis. These results show a possibility that HPV oral vaccine may be developed by expressing the HPV proteins in tomato or other vegetables.