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中华医学杂志(英文版) 2003年第7期000卷 133-139+70页

作者： reinhard h dennin,jianer wo Institut für Medizinische Mikrobiologie und hygiene Universitt zu Lübeck Lübeck Germany Institute of Infectious Diseases The First Affiliate hospital College of Medicine Zhejiang University hangzhou China

Objective To investigate a 272 base pair section of the 5' non coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (hCV) negative human subjects (not patients) Mothods This sequence section bears interest because ① it harbors several potential methylation (Cp rich) sites, and ② it represents the largest part of its internal ribosomal entry site A pre PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and hpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences Results The suspected hCV specific sequence was found in the DNA of each subject tested The pre PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena Conclusions The results provide formal proof that these hCV specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences

关键词： hCV sequences · 5’ NCR · IRES · human DNA · restriction endonucleases · methylation · epigenetic phenomena

human DNA contains sequences homologous to the 5'-non-coding region of hepatitis C virus: characterization with restriction endonucleases reveals individual varieties

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Chinese Medical Journal 2003年第7期116卷 1092-1098页

作者： reinhard h dennin jianer wo Institut für Medizinische Mikrobiologie und hygiene Universitt zu Lübeck Lübeck GermanyInstitute of Infectious Diseases The First Affiliate Hospital College of Medicine Zhejiang University Hangzhou China

关键词： hCV sequences · 5' NCR · IRES · human DNA · restriction endonucleases · methylation · epigenetic phenomena

Mutations in precore and core promoter region of hBV in patients with hepatic failure

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hepatobiliary & Pancreatic Diseases International 2002年第1期1卷 63-67页

作者： reinhard h. dennin Jian-Er wo Institute of Microbiology Luebeck Medical College Germany Zhejiang University School of Medicine Hangzhou 310003China

Objective: To clarify the association of hepatitis Bvirus mutants in precore and core promoter regions inpatients with hepatic failure and hBeAg ***: Precore and core promoter regions of 25hBV isolates from the patients with hepatic failurewere analyzed by polymerase chain reaction (PCR)direct sequencing ***: Precore G-to-A^(1896) mutants were identified in16 (64%) of the 25 isolates. The 'hot spot' mutationsat A-to-T^(1762) and G-to-A^(1764) were present together in19 (76%) of the 25 isolates, while C-to-T^(1653) and T-to-C^(1753) existed in a mutually exclusive manner andmore frequently in hepatic failure with liver cirrhosisgroup than in hepatic failure with chronic hepatitisgroup (100% vs 50%). Both A^(1896) and T^(1762)-A^(1764)could be found frequently in hBeAg-positive subjects(77.8% and 88.9%), whereas T^(1653)/C^(1753) was moreprevalent in anti-hBe-positive subjects than inhBeAg-positive subjects (93.8% vs 33.3%).Conclusions: The whole frequency of mutations in pre-core and core promoter gene will become more fre-quent as hBV infection is to be persistent. Mutationto T^(1653)/C^(1753) may be useful as a marker for hepaticfailure. It requires further study whether the mixedinfection of mutants and wilds will develop and affectthe condition of hBeAg in serum along the progres-sion of liver disease.

关键词： mutations gene hepatitis B virus

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DNA sequences homologous to hepatitis C virus(hCV) in the extrachromosomal circular DNA in peripheral blood mononuclear cells of hCV-negative subjects

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2019年第8期20卷 637-646页

作者： reinhard h. dennin Jian-Er wo Formerly Department of Infectious Diseases and Microbiology University of Lübeck University Hospital Schleswig-Holstein 23538 Lübeck Germany State Key Laboratory for Diagnosis and Treatment of Infectious Diseases Institute of Infectious Diseases the First Affiliated Hospital School of Medicine Zhejiang University Hangzhou 310003 China

Objective: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (hCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of hCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. Methods: home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. Results: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known hCV genotypes. In contrast, BLASTn alignment searches of published hCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with hCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. Conclusions: The experimental results confirm our assumption that parts of the hCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.

关键词： hepatitis C virus (hCV) 5'-Non-coding region (5'-NCR) human genome Extrachromosomal DNA Circular DNA Pattern of methylation

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DIFFERENT FACTORS AFFECTING ANTI-BODY RESPONSES IN MICE IMMUNIZED BY GENE RECOMBINANT OF hCV STRUCTURE REGION

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免疫学杂志 1999年第3期15卷 11-15页

作者：窦骏刘克洲陈智沃建尔何南祥刘勇章名太王信子徐陈槐浙江医科大学浙江杭州 310003

为寻求丙型肝炎病毒基因免疫的最佳动物实验方法，探讨不同处理方式对重组体ｐＣＤ－ＨＣＶ１诱发Ｂａｌｂ／ｃ小鼠产生抗体的影响。采用分子生物学技术构建了ＨＣＶ重组体，用不同免疫次数、途经、剂量和不同处理方式将其免疫小鼠。结... 详细信息

为寻求丙型肝炎病毒基因免疫的最佳动物实验方法，探讨不同处理方式对重组体ｐＣＤ－ＨＣＶ１诱发Ｂａｌｂ／ｃ小鼠产生抗体的影响。采用分子生物学技术构建了ＨＣＶ重组体，用不同免疫次数、途经、剂量和不同处理方式将其免疫小鼠。结果显示：重组体ｐＣＤ－ＨＣＶ１（１００μｇ／只／次，ｎ＝１２）经肌肉１次、２次、３次和４次免疫小鼠后，抗体水平分别为０．１８３±０．０６，０．４２８±０．０５，０．７０７±０．０８（ＯＤ４１０值，下同），其中４次免疫组抗体水平最高；小鼠经灌胃、腹腔注射、皮下注射和肌肉注射（１００μｇ／只×３次）不同途径分别免疫小鼠（ｎ＝６），抗体水平依次为０．１３８±０．０５，０．１７８±０．０７，０．２３３±０．０８和０．６９１±０．０５；以不同剂量（１０μｇ，５０μｇ，１００μｇ）分别免疫鼠（ｎ＝８），抗体水平依次为０．１１０±０．０９；０．３３０±０．０４和０．７００±０．０７，各组间差异均有显著性意义（Ｐ＜０．０１）；用普鲁卡因１００μｌ（０．０４ｍｇ）肌肉注射２４ｈ后，再肌肉、皮下注射ｐＣＤ－ＨＣＶ１，抗体水平比直接肌肉、皮下注射同剂量重组体明显增高。该研究结果为寻求ＨＣＶ－ＤＮＡ疫苗动物实验最佳免疫

关键词：重组体基因免疫 Balb/c小鼠抗体反应免疫方法