[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
Coagulation factor Ⅷ and antithrombin Ⅲ activity were detected in 15 health donors. It was found that antithrombin Ⅲ activity decreased obviously 12 h after blood drawing. It lost 56 % of the activity at the 3rd ...Coagulation factor Ⅷ and antithrombin Ⅲ activity were detected in 15 health donors. It was found that antithrombin Ⅲ activity decreased obviously 12 h after blood drawing. It lost 56 % of the activity at the 3rd day, and 70 % of the activity at the 7th day. FⅧ:c showed no obvious change after 24 h, until the 3rd day. It lost 40 %-60 % of the activity after 36 h and was reduced to the 30 % of the original activity at the 5th day. Our results suggested that at the 3rd day coagulation factor Ⅷ of bank stored blood can be used to replenish antithrombin Ⅲ, while bank stored blood in one day can be used to replenish FⅧ.展开更多
目的探索从Cohn氏法组分Ⅲ(组分Ⅲ)沉淀中纯化人凝血因子Ⅶ(FⅦ)的方法并对纯化产物进行鉴定。方法将组分Ⅲ沉淀溶解,经柠檬酸钡吸附、洗脱、硫酸铵沉淀、2次DEAE-Sepharose Fast Flow离子交换层析和Sephadex G-100凝胶过滤纯化人FⅦ。...目的探索从Cohn氏法组分Ⅲ(组分Ⅲ)沉淀中纯化人凝血因子Ⅶ(FⅦ)的方法并对纯化产物进行鉴定。方法将组分Ⅲ沉淀溶解,经柠檬酸钡吸附、洗脱、硫酸铵沉淀、2次DEAE-Sepharose Fast Flow离子交换层析和Sephadex G-100凝胶过滤纯化人FⅦ。结果从400g组分Ⅲ沉淀中最终得到10.1mg纯化的FⅦ,活性为1775.8U/mg,FⅦ被浓缩了近6000倍,总活性回收率为17.6%,SDS-PAGE电泳鉴定只有1条主条带。结论组分Ⅲ沉淀中有很高FⅧ活性;建立了从组分Ⅲ沉淀中纯化人FⅦ的方法。展开更多
BACKGROUND Multiple myeloma patients usually present with CRAB symptoms(hypercalcemia,renal disease,anemia and bone diseases)as initial manifestations.Bleeding symptoms are less common,most of which result from thromb...BACKGROUND Multiple myeloma patients usually present with CRAB symptoms(hypercalcemia,renal disease,anemia and bone diseases)as initial manifestations.Bleeding symptoms are less common,most of which result from thrombocytopenia or infiltration of plasmacytoma.Relatively,coagulopathy is not so common,especially isolated coagulopathy without CRAB manifestations,which is very rare.Herein,we report a 54-year old female who was hospitalized for intermittent and recurrent mild oral mucosal hemorrhage without other bleeding symptoms for almost one month or typical myeloma features.CASE SUMMARY Two months before admission,the patient underwent implantation of a permanent pacemaker due to sick sinus syndrome.Prothrombin time and activated partial thromboplastin time were significantly prolonged.Factor X deficiency was demonstrated to account for the coagulation dysfunction.An M protein peak was shown by serum protein electrophoresis.26.11%of abnormal plasma cells were detected in bone marrow by flow cytometry,expressing CD38,CD138,CD56 and intracellular immunoglobulin Kappa light chain.Bone marrow biopsy also proved the presence of abnormal plasma cells,but Congo red stain was negative.The patient was finally diagnosed with multiple myeloma IgA-κtype.A literature review indicated that factor X deficiency was highly related to amyloidosis.Before bleeding signs,the patient had cardiac arrhythmia,enlargement of the heart,and progressive heart failure;thus,cardiac amyloidosis was suspected.CONCLUSION Bleeding related to coagulation dysfunction is uncommon in multiple myeloma,especially as the initial manifestation.Amyloidosis is a well-recognized cause of isolated acquired factor X deficiency.展开更多
Anticoagulation factor Ⅰ (ACF Ⅰ) from the venom of Agkistrodon acutus prolonged plasma prothrombin time (PPT) with dose-dependent manner and exhibited marked anticoagulant activity only at the concentration higher t...Anticoagulation factor Ⅰ (ACF Ⅰ) from the venom of Agkistrodon acutus prolonged plasma prothrombin time (PPT) with dose-dependent manner and exhibited marked anticoagulant activity only at the concentration higher than its critical concentration (12 nmol/L). It was discovered that ACF Ⅰ formed a 1:1 complex with activated coagulation factor (FXa) in the presence of Ca2+ ions by the method of polyacrylamide gel electrophoresis. Both native ACF I and decalcified ACF Ⅰ failed to form complexes with FXa in the absence of Ca2+. Sr2+ ions were able to replace Ca2+ ions in the binding of ACF I to FXa, but both Ba2+ ions and Tb3+ ions were ineffective. ACF Ⅰ was a new member of the IX/X-bp family in the C-type lectin superfamily, and had a amino acid composition similar to the other members of this family. It was composed of 251 amino acid residues with a molecular weight of 29 603.6 u on non-reducing condition, determined by MALDI-TOF-MS, and a molecular weight of 14.7 ku on reducing condition,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.
文摘Coagulation factor Ⅷ and antithrombin Ⅲ activity were detected in 15 health donors. It was found that antithrombin Ⅲ activity decreased obviously 12 h after blood drawing. It lost 56 % of the activity at the 3rd day, and 70 % of the activity at the 7th day. FⅧ:c showed no obvious change after 24 h, until the 3rd day. It lost 40 %-60 % of the activity after 36 h and was reduced to the 30 % of the original activity at the 5th day. Our results suggested that at the 3rd day coagulation factor Ⅷ of bank stored blood can be used to replenish antithrombin Ⅲ, while bank stored blood in one day can be used to replenish FⅧ.
文摘目的探索从Cohn氏法组分Ⅲ(组分Ⅲ)沉淀中纯化人凝血因子Ⅶ(FⅦ)的方法并对纯化产物进行鉴定。方法将组分Ⅲ沉淀溶解,经柠檬酸钡吸附、洗脱、硫酸铵沉淀、2次DEAE-Sepharose Fast Flow离子交换层析和Sephadex G-100凝胶过滤纯化人FⅦ。结果从400g组分Ⅲ沉淀中最终得到10.1mg纯化的FⅦ,活性为1775.8U/mg,FⅦ被浓缩了近6000倍,总活性回收率为17.6%,SDS-PAGE电泳鉴定只有1条主条带。结论组分Ⅲ沉淀中有很高FⅧ活性;建立了从组分Ⅲ沉淀中纯化人FⅦ的方法。
文摘BACKGROUND Multiple myeloma patients usually present with CRAB symptoms(hypercalcemia,renal disease,anemia and bone diseases)as initial manifestations.Bleeding symptoms are less common,most of which result from thrombocytopenia or infiltration of plasmacytoma.Relatively,coagulopathy is not so common,especially isolated coagulopathy without CRAB manifestations,which is very rare.Herein,we report a 54-year old female who was hospitalized for intermittent and recurrent mild oral mucosal hemorrhage without other bleeding symptoms for almost one month or typical myeloma features.CASE SUMMARY Two months before admission,the patient underwent implantation of a permanent pacemaker due to sick sinus syndrome.Prothrombin time and activated partial thromboplastin time were significantly prolonged.Factor X deficiency was demonstrated to account for the coagulation dysfunction.An M protein peak was shown by serum protein electrophoresis.26.11%of abnormal plasma cells were detected in bone marrow by flow cytometry,expressing CD38,CD138,CD56 and intracellular immunoglobulin Kappa light chain.Bone marrow biopsy also proved the presence of abnormal plasma cells,but Congo red stain was negative.The patient was finally diagnosed with multiple myeloma IgA-κtype.A literature review indicated that factor X deficiency was highly related to amyloidosis.Before bleeding signs,the patient had cardiac arrhythmia,enlargement of the heart,and progressive heart failure;thus,cardiac amyloidosis was suspected.CONCLUSION Bleeding related to coagulation dysfunction is uncommon in multiple myeloma,especially as the initial manifestation.Amyloidosis is a well-recognized cause of isolated acquired factor X deficiency.
文摘Anticoagulation factor Ⅰ (ACF Ⅰ) from the venom of Agkistrodon acutus prolonged plasma prothrombin time (PPT) with dose-dependent manner and exhibited marked anticoagulant activity only at the concentration higher than its critical concentration (12 nmol/L). It was discovered that ACF Ⅰ formed a 1:1 complex with activated coagulation factor (FXa) in the presence of Ca2+ ions by the method of polyacrylamide gel electrophoresis. Both native ACF I and decalcified ACF Ⅰ failed to form complexes with FXa in the absence of Ca2+. Sr2+ ions were able to replace Ca2+ ions in the binding of ACF I to FXa, but both Ba2+ ions and Tb3+ ions were ineffective. ACF Ⅰ was a new member of the IX/X-bp family in the C-type lectin superfamily, and had a amino acid composition similar to the other members of this family. It was composed of 251 amino acid residues with a molecular weight of 29 603.6 u on non-reducing condition, determined by MALDI-TOF-MS, and a molecular weight of 14.7 ku on reducing condition,
