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乙型肝炎病毒特异性核酶与丁型肝炎病毒重组体的构建
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作者 温淑娟 祁学忠 +1 位作者 周荣 黄镇华 《第一军医大学学报》 CSCD 1999年第1期66-68,共3页
目的将重组丁型肝炎病毒基因组作为乙型肝炎病毒特异性核酶的载体,探讨解决核酶在实际应用中的保护性运载、靶向性和特异性问题。方法设计了乙型肝炎病毒特异性锤头型核酶结构,采用重组引物,结合“不对称PCR”和“MegaPri... 目的将重组丁型肝炎病毒基因组作为乙型肝炎病毒特异性核酶的载体,探讨解决核酶在实际应用中的保护性运载、靶向性和特异性问题。方法设计了乙型肝炎病毒特异性锤头型核酶结构,采用重组引物,结合“不对称PCR”和“MegaPrimerPCR”方法将所设计的锤头型核酶基因替代插入到了型肝炎病毒基因组5'端高变区,重组体经PCR初步鉴定后,克隆到T载体(pTAdv)T7启动子下游,经限制性内切酶酶切、序列测定分析。结果与结论证实该重组作为预期的乙型肝炎病毒特异性核酶-丁型肝炎病毒基因组重组体,为进一步的体外切割和细胞内与乙型肝炎病毒相互作用的研究提供了基础。 展开更多
关键词 丁型肝炎病毒 乙型肝炎病毒 核酶重组体 克隆
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丁型肝炎病毒载体携带核酶在小鼠体内抑制乙型肝炎病毒复制
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作者 李晓娟 《传染病网络动态》 2006年第7期108-109,共2页
目的:研究用丁型肝炎病毒(HDV)作为载体携带乙型肝炎病毒(HBV)特异性的锤头状核酶所构建的重组体,在细胞体系及转染动物模型中对HBV基因表达和复制的影响。方法:将HDV-核酶重组体和HBV的共表达质粒转染Huh-7细胞以分析HDV-核酶... 目的:研究用丁型肝炎病毒(HDV)作为载体携带乙型肝炎病毒(HBV)特异性的锤头状核酶所构建的重组体,在细胞体系及转染动物模型中对HBV基因表达和复制的影响。方法:将HDV-核酶重组体和HBV的共表达质粒转染Huh-7细胞以分析HDV-核酶重组体对HBV基因表达的影响;用小鼠尾静脉快速注射法将共表达质粒转染到小鼠体内,检测重组体在动物体内对HBV基因表达和复制的抑制作用。结果:转染细胞中,重组体对HBsAg的抑制与HDV重组位点和核酶靶位都有关;水压法注射的质粒在小鼠肝内得到表达,与对照相比重组HDV-核酶可有效抑制在肝和血清中HBV的基因表达以及复制。与细胞中的结果一致。结论:此项体内实验为进一步构建治疗性重组HDV病毒,发现靶向性抗病毒基因治疗手段奠定基础。 展开更多
关键词 乙型肝炎病毒复制 锤头状核酶 丁型肝炎病毒 内抑制 病毒载 乙型肝炎病毒(HBV) 小鼠 HUH-7细胞 核酶重组体 共表达质粒
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丁型肝炎
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《传染病网络动态》 2005年第9期112-112,共1页
丁型肝炎病毒基因组RNA包埋锤头状核酶的活性研究——李晓娟等(广东深圳市东湖医院、深圳市肝病研究所518020):《中华实验和临床病毒学杂志》,2005,19(1):12-15[目的:探讨用丁型肝炎病毒(HDV)基因组来包埋HBV靶向性核酶对核... 丁型肝炎病毒基因组RNA包埋锤头状核酶的活性研究——李晓娟等(广东深圳市东湖医院、深圳市肝病研究所518020):《中华实验和临床病毒学杂志》,2005,19(1):12-15[目的:探讨用丁型肝炎病毒(HDV)基因组来包埋HBV靶向性核酶对核酶体内外活性产生的影响。方法:用和HBV靶基因体外转录产物在不同反应条件下温育对HDV-核酶重组体的体外切割活性定量分析: 展开更多
关键词 丁型肝炎病毒 基因组RNA 外切割活性 锤头状核酶 临床病毒学 内外活性 核酶重组体 活性研究 定量分析
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Expression and Characterization of the Recombinant Human FLT-3 Ligand Extracellular Domain in Pichia Pastoris
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作者 Zongtang Huang Xishan Hao 《Chinese Journal of Clinical Oncology》 CSCD 2006年第6期400-407,共8页
OBJECTIVE The FLT-3 ligand (fms-like tyrosine kinase receptor-3 ligand, FL) is a recently described growth factor affecting early hematopoietic progenitor cells. The FL plays a key role in the growth and differentia... OBJECTIVE The FLT-3 ligand (fms-like tyrosine kinase receptor-3 ligand, FL) is a recently described growth factor affecting early hematopoietic progenitor cells. The FL plays a key role in the growth and differentiation of primitive hematopoietic cells. To yield a high-level of recombinant human FL protein, a recembinant Pichia Pastoris (P. pastoris)strain was constructed. METHODS An artificial expression frame, with the same encoding protein sequence for the FL extracellular domain cDNA, was synthesized by using favored genetic codons of P. pastoris. P. pastoris strain KM71 cells were transformed with the endonuclease Bgl II linearized recombined plasmid, pPIC9K-FL. The plasmid then was linerized in the 5'AOX1 site and integrated into the yeast KM71 genome. KM71 was transformed with pPIC9K plasmids as a control for the production of recombinant protein. Southern blotting and Northern blotting tests were used to screen the genotype of the recombined strain. Biological activity was demonstrated in vitro with culturing of CD34+cells. RESULTS The recombinant human FL protein expressed into the yeast culture supertant was identified on the basis of its molecular weight and Western blotting analysis. Numerous bands were observed in the 10-100 kDa molecular mass range. SDS-PAGE showed that the expressed product, a 20 kDa protein, was secreted into the medium in the form of a soluble molecule. Western-blot analyses showed good antigenicity and specificity against polyclonal antibodies. A sharp band and a smeared band were observed at a molecular mass of approximately 20 kDa by Western blotting. The recombinant human FL protein was the major protein component observed in the culture supernatant. The highest yield (108 mg/L) was obtained when expression was induced with 0.5% methanol for 96 h. Deglycosylation with PNGase F resulted in a decrease in apparent molecular mass from 20 kDa to 18kDa forming three bands all of which were also detected by rabbit anti-FL antibodies, Culturing of CD34+ cells in the presence of KM71pPIC9K-FL over 7 days increased 2.9 fold, while in the control group they increased only 1,5 fold. The biological assay showed that the expressed product could stimulate the proliferation of CD34+ hematopoietic cells, CONCLUSION We demonstrated that human FL was secreted into the culture supernatant from P. pastoris, and that this yeast strain was a preferred host for recombinant human FL gene expression. This recombinant strain can provide a convenient process for pharmaceutical application. 展开更多
关键词 EXPRESSION RECOMBINANT human FLT-3 ligand extmcellular domain Pichia pastoris.
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