[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
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
目的探讨人纤维蛋白原(FIB)在急诊创伤性凝血病中的应用及危险因素分析。方法选取2018年1月至2019年12月联保部队第九O八医院收治的100例严重外伤所致创伤性凝血病患者作为观察组,按照随机数字表法分为A组与B组,每组50例;另选取50例急...目的探讨人纤维蛋白原(FIB)在急诊创伤性凝血病中的应用及危险因素分析。方法选取2018年1月至2019年12月联保部队第九O八医院收治的100例严重外伤所致创伤性凝血病患者作为观察组,按照随机数字表法分为A组与B组,每组50例;另选取50例急诊创伤性非凝血病患者作为对照组。A组给予急诊创伤性常规治疗,B组在A组基础上给予人FIB治疗,对照组治疗方法同A组。比较A组和B组治疗前后凝血功能指标[活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、FIB、D-二聚体(D-D)],24 h新鲜冰冻血浆(FFP)输注量、24 h浓缩红细胞(CRBC)输注量、ICU住院时间及病死率。采用多因素Logistic回归分析影响创伤性凝血病的危险因素。结果治疗后,两组APTT、PT均短于治疗前,FIB水平均高于治疗前,D-D水平均低于治疗前,且B组APTT、PT均短于A组,FIB水平高于A组,D-D水平低于A组,差异有统计学意义(P<0.001)。B组24 h FFP输注量、24 h CRBC输注量均少于A组,病死率低于A组,ICU住院时间短于A组,差异有统计学意义(P<0.05)。观察组APTT、PT均长于对照组,国际标准化比值(INR)、损伤严重程度评分(ISS)、急性生理学和慢性健康状况评价(APACHE)Ⅱ评分、格拉斯哥昏迷(GCS)评分及碱缺失(BD)≥6 mol/L、合并重型颅脑损伤(STBI)、体温<35℃比例均高于对照组,24 h FFP、24 h CRBC输注量均多于对照组,血红蛋白(Hb)水平、血小板计数(PLT)均低于对照组,差异有统计学意义(P<0.05)。多因素Logistic回归分析显示,PLT低、GCS评分<6分、BD≥6 mol/L、合并STBI是创伤性凝血病发生的独立危险因素(P<0.05)。结论人FIB可改善急性创伤性凝血病凝血功能,减少血液输注量,缩短ICU住院时间及降低病死率,临床疗效显著,PLT低水平、GCS评分<6分、BD≥6 mol/L、合并STBI是创伤性凝血病发生的独立危险因素。展开更多
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
基金Supported by Anhui Provincial Natural Science Foundation of China(2008085MC65)Natural Science Foundation of Anhui Higher Education Institutions of China(KJ2021A0922)+1 种基金China Postdoctoral Science Foundation(2020T130117ZX,2020M671914)Research Activities of Postdoctoral Researchers Foundation of Anhui Province,China(2020B470)。
文摘[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.
基金Supported by FJIRSM (SZD08003)the National Natural Science Foundation of China(30811130467,30625011,30770429)Chinese Academy of Sciences (KSCX2-YW-R-082)
文摘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.
文摘目的探讨人纤维蛋白原(FIB)在急诊创伤性凝血病中的应用及危险因素分析。方法选取2018年1月至2019年12月联保部队第九O八医院收治的100例严重外伤所致创伤性凝血病患者作为观察组,按照随机数字表法分为A组与B组,每组50例;另选取50例急诊创伤性非凝血病患者作为对照组。A组给予急诊创伤性常规治疗,B组在A组基础上给予人FIB治疗,对照组治疗方法同A组。比较A组和B组治疗前后凝血功能指标[活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、FIB、D-二聚体(D-D)],24 h新鲜冰冻血浆(FFP)输注量、24 h浓缩红细胞(CRBC)输注量、ICU住院时间及病死率。采用多因素Logistic回归分析影响创伤性凝血病的危险因素。结果治疗后,两组APTT、PT均短于治疗前,FIB水平均高于治疗前,D-D水平均低于治疗前,且B组APTT、PT均短于A组,FIB水平高于A组,D-D水平低于A组,差异有统计学意义(P<0.001)。B组24 h FFP输注量、24 h CRBC输注量均少于A组,病死率低于A组,ICU住院时间短于A组,差异有统计学意义(P<0.05)。观察组APTT、PT均长于对照组,国际标准化比值(INR)、损伤严重程度评分(ISS)、急性生理学和慢性健康状况评价(APACHE)Ⅱ评分、格拉斯哥昏迷(GCS)评分及碱缺失(BD)≥6 mol/L、合并重型颅脑损伤(STBI)、体温<35℃比例均高于对照组,24 h FFP、24 h CRBC输注量均多于对照组,血红蛋白(Hb)水平、血小板计数(PLT)均低于对照组,差异有统计学意义(P<0.05)。多因素Logistic回归分析显示,PLT低、GCS评分<6分、BD≥6 mol/L、合并STBI是创伤性凝血病发生的独立危险因素(P<0.05)。结论人FIB可改善急性创伤性凝血病凝血功能,减少血液输注量,缩短ICU住院时间及降低病死率,临床疗效显著,PLT低水平、GCS评分<6分、BD≥6 mol/L、合并STBI是创伤性凝血病发生的独立危险因素。
文摘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-
