Objective: To explore high-yield secretory expression of recombinant mouse coagulation factor Ⅶ (rmF Ⅶ ) protein in Pichia pastoris (P. pastoris). Methods: The fragment of mF Ⅶ cDNA was amplified by PCR from a pcDN...Objective: To explore high-yield secretory expression of recombinant mouse coagulation factor Ⅶ (rmF Ⅶ ) protein in Pichia pastoris (P. pastoris). Methods: The fragment of mF Ⅶ cDNA was amplified by PCR from a pcDNA3-mFⅦ plasmid. Then the cDNA fragment was subcloned into α-factor secretion signal open reading frame of pPIC9K secretory expression vector. The mutagenesis of mF Ⅶ was performed by Site-Direct Mutation and then verified by DNA sequencing. The yeast expression vector of rmF Ⅶ, named as pPIC9K-rmFⅦ, was linearized with Sac I and transferred into GS115 strains(his-Mut+)by electroporation. The recombinants were identified by direct PCR and selection on MM and MD plates. rmF Ⅶ was expressed in recombinant strains (his+Mut+) for 4 d. The expression level and activation of rmF Ⅶ in the BMMY medium were detected by SDS-PAGE and Western blot respectively. Results:pPIC9K-rmFⅦ was constructed and transferred to GS115 strains successfully. 48-hour post induction by methanol rmFⅦ protein was secreted into the culture supernatant. The molecular weight of the expressed products was shown to be about 46 kD by SDS-PAGE analysis. Western blot showed that the expressed rmF Ⅶ exhibited specificity and antigenicity. Conclusion: Since mFⅦ is considered as a tumor-targeting molecule , this study may provide a basis for further anti-tumor strategy on rmFⅦ.展开更多
BACKGROUND： In adults, vitamin K-dependent coagulation factor deficiency （VKCFD） increases in the recent years. We treated a VKCFD patient with subarachnoid hemorrhage, with favorable outcomes.METHODS： A 19-year-o...BACKGROUND： In adults, vitamin K-dependent coagulation factor deficiency （VKCFD） increases in the recent years. We treated a VKCFD patient with subarachnoid hemorrhage, with favorable outcomes.METHODS： A 19-year-old male student with VKCFD was treated at our hospital. The initial treatment was injection of a large dose of vitamin K and fresh plasma, and then with oral high dose of vitamin K4.RESULTS： At 4 weeks after admission, the focus of hemorrhage subsided, neurological examination was normal, and the patient was discharged.CONCLUSIONS： VKCFD is rare and its diagnosis should be based on the history of the patient and the results of laboratory examinations. A large dose of vitamin K is the fi rst choice of treatment.展开更多
Objective: To construct and apply a replacement targeting vector for mouse coagulation factor IX(mFIX) gene in embryonic stem (ES) cells. Methods: Based on the cloning and structural analysis of the genomic DNA fragme...Objective: To construct and apply a replacement targeting vector for mouse coagulation factor IX(mFIX) gene in embryonic stem (ES) cells. Methods: Based on the cloning and structural analysis of the genomic DNA fragment of coagulation factor IX gene from 129Sv mouse genomic DNA A phage library, PMFIXDEL plasmid was designed with positive-negative-selection (PNS) strategy, and constructed with commonmolecular cloning techniques. Structure of PMFIXDEL was identified by PCR and restriction analysis. Afterelectroporation with the linearized PMFIXDEL DNA, transfected ES cells were cultured in G418/GANC drugselection medium. The recombination efficacy of this vector was tested. Results: The main components ofPMFIXDEL were two copies of negative selection gene (HSV-tk expression cassette), a positive selectiongene (Neo expression cassette), long and short homologous fragments and plasmid backbone. The introduction of negative selection gene (HSV-tk ) into the construct resulted in 24-fold increase of selection.Conclusion: An effective replacement vector for mFIX gene targeting was constructed and applied in ES cell.展开更多
A comprehensive method was applied to evaluate the anticoagulant activity of a novel anticoagulant peptide(NAESLRK)derived from oyster(Crassostrea gigas).The anticoagulant peptide drastically reduced the extrinsic clo...A comprehensive method was applied to evaluate the anticoagulant activity of a novel anticoagulant peptide(NAESLRK)derived from oyster(Crassostrea gigas).The anticoagulant peptide drastically reduced the extrinsic clotting activity and also impaired the intrinsic clotting activity slightly.Consistent with clotting data,the thrombin peak height was reduced to 84.7 nmol/L from 123.4 nmol/L,and thrombin generation time was delayed to 4.67 min from 4.42 min when the extrinsic trigger was applied.The inhibitory kinetics of FⅪa,FⅨa,FⅩa,FⅡa,and APC in a purified component system rationally explained the reduction of extrinsic clotting activity and impairment of thrombin generation.Besides the inhibition of FⅩa and FⅡa activity,the activation processes of FⅩand FⅡby intrinsic/extrinsic tenase complex and prothrombinase were also damaged.The anticoagulant activity in the plasma system was the result of comprehensive inhibition of various factors.The research provided a novel method for anticoagulant evaluation and inhibitory mechanism of bioactive peptides from food products.展开更多
objective:To study systematically the abilities of skeletal muscle cells in synthesizing,processing and secreting recombinant proteins in vitro.Methods:Primary murine myoblasts from SCID mice (SCID-MB) and C2C12,a mus...objective:To study systematically the abilities of skeletal muscle cells in synthesizing,processing and secreting recombinant proteins in vitro.Methods:Primary murine myoblasts from SCID mice (SCID-MB) and C2C12,a muse myoblast cell line,were trans fected with muscle specific (pdLMe4bAh ff m) or non-specif ic (LIXSN) vectors encoding human factor IX (FIX). The capacities of the transgenic cells in producing re combinant FIX were compared in the levels of intracellular and secreted FIX protein as well as FIX mRNA in primary muscle cells and C2C12 cells. Results:Both primary muscle cells and C2C12 cells can efficiently trans late and process FIX protein. Myotubes derived from the SCID-MB produced 1 800~2 000 ng FIX/106 cells/ 24 h in culture with specific activity of 95% ~ 103%. C2C12 cells can synthesize and process recombinant FIX as efficiently as primary muscle cells,but their secretion ability is less efficient in comparison with primary cells. Conclusion:This observation indicates that primary cells should be used in gene therapy related research projects and care should be taken while explaining the results derived from established cell lines.展开更多
基金Supported by the grants from Academician Foundation of Chongqing (2004BC5006)
文摘Objective: To explore high-yield secretory expression of recombinant mouse coagulation factor Ⅶ (rmF Ⅶ ) protein in Pichia pastoris (P. pastoris). Methods: The fragment of mF Ⅶ cDNA was amplified by PCR from a pcDNA3-mFⅦ plasmid. Then the cDNA fragment was subcloned into α-factor secretion signal open reading frame of pPIC9K secretory expression vector. The mutagenesis of mF Ⅶ was performed by Site-Direct Mutation and then verified by DNA sequencing. The yeast expression vector of rmF Ⅶ, named as pPIC9K-rmFⅦ, was linearized with Sac I and transferred into GS115 strains(his-Mut+)by electroporation. The recombinants were identified by direct PCR and selection on MM and MD plates. rmF Ⅶ was expressed in recombinant strains (his+Mut+) for 4 d. The expression level and activation of rmF Ⅶ in the BMMY medium were detected by SDS-PAGE and Western blot respectively. Results:pPIC9K-rmFⅦ was constructed and transferred to GS115 strains successfully. 48-hour post induction by methanol rmFⅦ protein was secreted into the culture supernatant. The molecular weight of the expressed products was shown to be about 46 kD by SDS-PAGE analysis. Western blot showed that the expressed rmF Ⅶ exhibited specificity and antigenicity. Conclusion: Since mFⅦ is considered as a tumor-targeting molecule , this study may provide a basis for further anti-tumor strategy on rmFⅦ.
文摘BACKGROUND： In adults, vitamin K-dependent coagulation factor deficiency （VKCFD） increases in the recent years. We treated a VKCFD patient with subarachnoid hemorrhage, with favorable outcomes.METHODS： A 19-year-old male student with VKCFD was treated at our hospital. The initial treatment was injection of a large dose of vitamin K and fresh plasma, and then with oral high dose of vitamin K4.RESULTS： At 4 weeks after admission, the focus of hemorrhage subsided, neurological examination was normal, and the patient was discharged.CONCLUSIONS： VKCFD is rare and its diagnosis should be based on the history of the patient and the results of laboratory examinations. A large dose of vitamin K is the fi rst choice of treatment.
文摘Objective: To construct and apply a replacement targeting vector for mouse coagulation factor IX(mFIX) gene in embryonic stem (ES) cells. Methods: Based on the cloning and structural analysis of the genomic DNA fragment of coagulation factor IX gene from 129Sv mouse genomic DNA A phage library, PMFIXDEL plasmid was designed with positive-negative-selection (PNS) strategy, and constructed with commonmolecular cloning techniques. Structure of PMFIXDEL was identified by PCR and restriction analysis. Afterelectroporation with the linearized PMFIXDEL DNA, transfected ES cells were cultured in G418/GANC drugselection medium. The recombination efficacy of this vector was tested. Results: The main components ofPMFIXDEL were two copies of negative selection gene (HSV-tk expression cassette), a positive selectiongene (Neo expression cassette), long and short homologous fragments and plasmid backbone. The introduction of negative selection gene (HSV-tk ) into the construct resulted in 24-fold increase of selection.Conclusion: An effective replacement vector for mFIX gene targeting was constructed and applied in ES cell.
基金financially supported by the National Natural Science Foundation of China(31771926)the State Key Research and Development Plan“Modern Food Processing and Food Storage and Transportation Technology and Equipment”(No.2017YFD0400201)。
文摘A comprehensive method was applied to evaluate the anticoagulant activity of a novel anticoagulant peptide(NAESLRK)derived from oyster(Crassostrea gigas).The anticoagulant peptide drastically reduced the extrinsic clotting activity and also impaired the intrinsic clotting activity slightly.Consistent with clotting data,the thrombin peak height was reduced to 84.7 nmol/L from 123.4 nmol/L,and thrombin generation time was delayed to 4.67 min from 4.42 min when the extrinsic trigger was applied.The inhibitory kinetics of FⅪa,FⅨa,FⅩa,FⅡa,and APC in a purified component system rationally explained the reduction of extrinsic clotting activity and impairment of thrombin generation.Besides the inhibition of FⅩa and FⅡa activity,the activation processes of FⅩand FⅡby intrinsic/extrinsic tenase complex and prothrombinase were also damaged.The anticoagulant activity in the plasma system was the result of comprehensive inhibition of various factors.The research provided a novel method for anticoagulant evaluation and inhibitory mechanism of bioactive peptides from food products.
文摘objective:To study systematically the abilities of skeletal muscle cells in synthesizing,processing and secreting recombinant proteins in vitro.Methods:Primary murine myoblasts from SCID mice (SCID-MB) and C2C12,a muse myoblast cell line,were trans fected with muscle specific (pdLMe4bAh ff m) or non-specif ic (LIXSN) vectors encoding human factor IX (FIX). The capacities of the transgenic cells in producing re combinant FIX were compared in the levels of intracellular and secreted FIX protein as well as FIX mRNA in primary muscle cells and C2C12 cells. Results:Both primary muscle cells and C2C12 cells can efficiently trans late and process FIX protein. Myotubes derived from the SCID-MB produced 1 800~2 000 ng FIX/106 cells/ 24 h in culture with specific activity of 95% ~ 103%. C2C12 cells can synthesize and process recombinant FIX as efficiently as primary muscle cells,but their secretion ability is less efficient in comparison with primary cells. Conclusion:This observation indicates that primary cells should be used in gene therapy related research projects and care should be taken while explaining the results derived from established cell lines.
