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Expression and Purification of Human Coagulation Factor X in Mammalian CHO-DG44 Cells
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作者 Jinwu CHEN Yi LI +4 位作者 Mei LIU Sainan WANG Zilong XIAO Junjie XIA Lulu QI 《Agricultural Biotechnology》 CAS 2023年第3期50-54,共5页
[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different ... [Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX. 展开更多
关键词 Recombinant human coagulation factor X(rhFX) Eukaryotic expression MTX Affinity chromatography
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Preparation of monoclon alantibodies to human coagulation factorⅩ
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《中国输血杂志》 CAS CSCD 2001年第S1期407-,共1页
关键词 Preparation of monoclon alantibodies to human coagulation factor
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Molecular mechanism of microRNA125 regulating human coagulation factor IX gene with nonsense mutation
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作者 王刚 《China Medical Abstracts(Internal Medicine)》 2016年第3期182-,共1页
Objective To construct human coagulation factorⅨmini-gene(Mini-h F9)and some nonsense mutants,detect the levels of the Mini-h F9 mRNA,and analyze the molecular mechanism of microRNA125 regulating F9gene with nonsense... Objective To construct human coagulation factorⅨmini-gene(Mini-h F9)and some nonsense mutants,detect the levels of the Mini-h F9 mRNA,and analyze the molecular mechanism of microRNA125 regulating F9gene with nonsense mutation.Methods Three nonsense mutants were obtained by using PCR mutagenesis to ana- 展开更多
关键词 GENE Molecular mechanism of microRNA125 regulating human coagulation factor IX gene with nonsense mutation mRNA MICRORNA
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Preparation and Structure of a New Coagulation Factor XI Catalytic Domain for Drug Discovery 被引量:1
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作者 江龙光 袁彩 +4 位作者 陈宏炜 王宇 赵宝玉 张旭 黄明东 《Chinese Journal of Structural Chemistry》 SCIE CAS CSCD 2011年第7期1021-1029,共9页
Human blood coagulation factor XI (FXI) is a key enzyme in the amplification phase of blood coagulation cascade, and is recognized as an important target for anti-coagulant development in recent years. We designed a... Human blood coagulation factor XI (FXI) is a key enzyme in the amplification phase of blood coagulation cascade, and is recognized as an important target for anti-coagulant development in recent years. We designed a new mutant form of FXIa catalytic domain rhFXI370-607 (N73Q-N113Q-C123S), and report here the facile preparation, protein crystallization, and crystal structure of this protein. We highlight a few unique structural features of FXIa after comparison with the trypsin family serine proteases at sequence and structural levels. This work provides a foundation to develop new small molecular FXIa inhibitors with increased potency and specificity. 展开更多
关键词 human blood coagulation factor XI crystal structure serine proteases INHIBITORS
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