10C12 is an anticoagulant antibody identified from a phage display single-chain Fv human antibody library. It can be directed at the calcium-stabilized Gla domain of Factor-IX, an important coagulation factor in intri...10C12 is an anticoagulant antibody identified from a phage display single-chain Fv human antibody library. It can be directed at the calcium-stabilized Gla domain of Factor-IX, an important coagulation factor in intrinsic pathway of blood coagulation cascade, and interfere with membrane anchoring of Factor IX, thus inhibiting blood coagulation function. 10C12 has been demonstrated as an effective anti-coagulant in attenuating thrombosis in several different animal models. Here, we report the crystal structure of the Fab fragment of 10C12. The crystal contains two Fab molecules in the asymmetric unit with identical conformation, forming a lattice with large cavities. In addition, comparison of this free Fab with the antigen-bound structure of 10C12 shows no change in CDR conformations and the relative disposition of the variable subunits of H and L chains, suggesting the rigid conformation of this 10C12 Fab and a lock-and-key mechanism of antibody-antigen recognition for 10C12.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
DNA sequence of 2.2kb of factor IX gene from each of 27 cases of Hemophilia B patients living in Jiangsu, Hubei, Shandong, Guangdong, Fujian and Ningxia provinces was studied by using the PCR (Polymerase Chain Reactio...DNA sequence of 2.2kb of factor IX gene from each of 27 cases of Hemophilia B patients living in Jiangsu, Hubei, Shandong, Guangdong, Fujian and Ningxia provinces was studied by using the PCR (Polymerase Chain Reaction) and GAWTS (Genomic Amplification with Transcript Sequencing) techniques. Twenty different mutations were found in 21 patients. Twelve of them were not reported before. Eight occurred in CpG dinucleotide (38%) that was a hot spot of germ line mutations in Caucasian. No geographic specificity was found between 6 provinces. Six carriers were identified. Some advantages of the methodology were discussed.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
This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous nitron 1 sequence. hFIX minigene was obtained with middle sequence truncated nitron 1 inserted into the relative site ...This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous nitron 1 sequence. hFIX minigene was obtained with middle sequence truncated nitron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i’IX, retroviral vector GINaCi’IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKG5i’IX, 151 "g/106 cells/24h; PA317/GINaCi’IX, 308ng/106 cells/24 h; C2C12/G1 NaCi’IX, 186 ng/106 cells/24 h; RSF/GINaCi’IX, 1929 ng/106 cells/24 h; HSF/GlNaCi’IX, 1646 ng/106 cells/ 24 h. These results indicated that hFIX minigene with nitron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from nitron splicing during viral production, a retroviral vector GlNaCi’IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/106 cells/24 h with 79% of bioactivity. PCR detection of HT/GlNaCi’IXR cells infected with PA317/ClNaCi’IXR supernatant confirmed the existence of nitron 1 sequence. These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene Human factor IX expression with nitron transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.展开更多
基金the grants from Fujian Explore Grant for Young Scientists (2007J0294)CAS (KSCX2-YW-R-082)+1 种基金NNSFC (30430190, 30625011)MOST (2007CB914304 and 2006AA02A313)
文摘10C12 is an anticoagulant antibody identified from a phage display single-chain Fv human antibody library. It can be directed at the calcium-stabilized Gla domain of Factor-IX, an important coagulation factor in intrinsic pathway of blood coagulation cascade, and interfere with membrane anchoring of Factor IX, thus inhibiting blood coagulation function. 10C12 has been demonstrated as an effective anti-coagulant in attenuating thrombosis in several different animal models. Here, we report the crystal structure of the Fab fragment of 10C12. The crystal contains two Fab molecules in the asymmetric unit with identical conformation, forming a lattice with large cavities. In addition, comparison of this free Fab with the antigen-bound structure of 10C12 shows no change in CDR conformations and the relative disposition of the variable subunits of H and L chains, suggesting the rigid conformation of this 10C12 Fab and a lock-and-key mechanism of antibody-antigen recognition for 10C12.
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.
基金the High Technology Research and Development Programme of China and USA Grant ROIHL 39762
文摘DNA sequence of 2.2kb of factor IX gene from each of 27 cases of Hemophilia B patients living in Jiangsu, Hubei, Shandong, Guangdong, Fujian and Ningxia provinces was studied by using the PCR (Polymerase Chain Reaction) and GAWTS (Genomic Amplification with Transcript Sequencing) techniques. Twenty different mutations were found in 21 patients. Twelve of them were not reported before. Eight occurred in CpG dinucleotide (38%) that was a hot spot of germ line mutations in Caucasian. No geographic specificity was found between 6 provinces. Six carriers were identified. Some advantages of the methodology were discussed.
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.
文摘This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous nitron 1 sequence. hFIX minigene was obtained with middle sequence truncated nitron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i’IX, retroviral vector GINaCi’IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKG5i’IX, 151 "g/106 cells/24h; PA317/GINaCi’IX, 308ng/106 cells/24 h; C2C12/G1 NaCi’IX, 186 ng/106 cells/24 h; RSF/GINaCi’IX, 1929 ng/106 cells/24 h; HSF/GlNaCi’IX, 1646 ng/106 cells/ 24 h. These results indicated that hFIX minigene with nitron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from nitron splicing during viral production, a retroviral vector GlNaCi’IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/106 cells/24 h with 79% of bioactivity. PCR detection of HT/GlNaCi’IXR cells infected with PA317/ClNaCi’IXR supernatant confirmed the existence of nitron 1 sequence. These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene Human factor IX expression with nitron transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.
