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
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
Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β\|casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were tran...Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β\|casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and LacZ gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector.展开更多
目的了解不同浓度水平的肝素、抗凝血酶Ⅲ(ATⅢ)或肝素/ATⅢ(1∶1混)对人凝血因子Ⅸ(FⅨ)类制品中FⅨ效价检测的影响。方法向FⅨ或人凝血酶原复合物(PCC)样品中加入不同浓度水平的肝素或肝素/ATⅢ来制备每1 IU FⅨ含0、0.1、0.3、0.5、...目的了解不同浓度水平的肝素、抗凝血酶Ⅲ(ATⅢ)或肝素/ATⅢ(1∶1混)对人凝血因子Ⅸ(FⅨ)类制品中FⅨ效价检测的影响。方法向FⅨ或人凝血酶原复合物(PCC)样品中加入不同浓度水平的肝素或肝素/ATⅢ来制备每1 IU FⅨ含0、0.1、0.3、0.5、0.8、1、2、4 IU的肝素或肝素/ATⅢ的样品以及向FⅨ或PCC样品中单独加入ATⅢ时制备每1 IU FⅨ中含0、0.1、0.5、1 IU ATⅢ的样品后,采用一期凝固法测定所制备样品中的FⅨ效价;取适量制备的含不同肝素浓度的样品,分别加入相应量的硫酸鱼精蛋白中和样品中的肝素后再次测定FⅨ效价。观察并分析不同浓度水平肝素、ATⅢ或肝素/ATⅢ对FⅨ效价检测的影响。结果当样品中每1 IU FⅨ的肝素或肝素/ATⅢ含量为0、0.1、0.3、0.5 IU时,FⅨ效价检测结果较一致;当肝素或肝素/ATⅢ含量为0.8、1、2、4 IU时,FⅨ效价检测结果均低于不含肝素样品的检测结果;ATⅢ含量为0、0.1、0.5、1 IU时,样品中FⅨ效价检测一致。结论当FⅨ或PCC制品中每1 IU FⅨ含肝素或肝素/ATⅢ的量≤0.5 IU时,对FⅨ效价的检测结果无影响,可省略加入硫酸鱼精蛋白中和肝素的步骤;>0.5 IU时,宜加入硫酸鱼精蛋白中和肝素后再检测FⅨ效价。展开更多
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.
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-
文摘Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β\|casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and LacZ gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector.
文摘目的了解不同浓度水平的肝素、抗凝血酶Ⅲ(ATⅢ)或肝素/ATⅢ(1∶1混)对人凝血因子Ⅸ(FⅨ)类制品中FⅨ效价检测的影响。方法向FⅨ或人凝血酶原复合物(PCC)样品中加入不同浓度水平的肝素或肝素/ATⅢ来制备每1 IU FⅨ含0、0.1、0.3、0.5、0.8、1、2、4 IU的肝素或肝素/ATⅢ的样品以及向FⅨ或PCC样品中单独加入ATⅢ时制备每1 IU FⅨ中含0、0.1、0.5、1 IU ATⅢ的样品后,采用一期凝固法测定所制备样品中的FⅨ效价;取适量制备的含不同肝素浓度的样品,分别加入相应量的硫酸鱼精蛋白中和样品中的肝素后再次测定FⅨ效价。观察并分析不同浓度水平肝素、ATⅢ或肝素/ATⅢ对FⅨ效价检测的影响。结果当样品中每1 IU FⅨ的肝素或肝素/ATⅢ含量为0、0.1、0.3、0.5 IU时,FⅨ效价检测结果较一致;当肝素或肝素/ATⅢ含量为0.8、1、2、4 IU时,FⅨ效价检测结果均低于不含肝素样品的检测结果;ATⅢ含量为0、0.1、0.5、1 IU时,样品中FⅨ效价检测一致。结论当FⅨ或PCC制品中每1 IU FⅨ含肝素或肝素/ATⅢ的量≤0.5 IU时,对FⅨ效价的检测结果无影响,可省略加入硫酸鱼精蛋白中和肝素的步骤;>0.5 IU时,宜加入硫酸鱼精蛋白中和肝素后再检测FⅨ效价。
