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
[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
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.
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.
