This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a s...This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp).展开更多
目的:建立重组 hPK-5质粒 DNA 基因治疗制剂的质量标准和检测方法。方法:采用限制性酶切图谱分析鉴定质粒DNA 结构和 PCR 法鉴定插入的 hPK-5基因作鉴别试验;采用分光光度法测定质粒 DNA 含量和 A_(260)/A_(280)比值;采用琼脂糖凝胶电...目的:建立重组 hPK-5质粒 DNA 基因治疗制剂的质量标准和检测方法。方法:采用限制性酶切图谱分析鉴定质粒DNA 结构和 PCR 法鉴定插入的 hPK-5基因作鉴别试验;采用分光光度法测定质粒 DNA 含量和 A_(260)/A_(280)比值;采用琼脂糖凝胶电泳法分析质粒 DNA 的超螺旋构象比例和残留 RNA 含量;采用阴离子交换色谱法分析 HPLC 纯度;采用 ELISA 法测定hPK～5蛋白的表达量;采用内皮细胞迁移法测定 hPK-5蛋白的生物学活性。结果:重组质粒 DNA 的限制性酶切片段大小与理论值一致;插入基因 PCR 扩增片段大小与理论值相符;质粒 DNA 含量为1.12 mg·mL^(-1);A_(260)/A_(280)比值为1.83;琼脂糖凝胶电泳法测定超螺旋构象质粒的比例为95.4%;HPLC 测定超螺旋质粒 DNA 占93.8%,开环质粒 DNA 占6.2%,琼脂糖凝胶电泳法测定木检出 RNA 残留;没有检出其他杂质;以2μg重组 hPK-5质粒转染5×10~5Hela 细胞48 h 后,2 mL 培养上清中 hPK-5蛋白浓度为109 ng·mL^(-1);以培养上清进行内皮细胞迁移试验,转染重组 hPK-5质粒 DNA 组迁移的内皮细胞数明显少于对照绀(P<0.05)。其他项目均符合相应的规定。结论:建立了重组 hPK-5质粒 DNA 基因治疗制剂的检定方法及其质量标准,为有效控制该类产品的质量打下基础。展开更多
目的 :构建人血管内皮生长因子 ( VEGF) c DNA逆转录病毒表达质粒。方法 :采用 PCR方法从 pc DNA- VEGF165质粒中扩增 VEGF165基因 ,在序列两端分别引入 Eco R 和 Bam H 限制性内切酶识别位点 ,并克隆到逆转录病毒表达载体 p LXSN质粒...目的 :构建人血管内皮生长因子 ( VEGF) c DNA逆转录病毒表达质粒。方法 :采用 PCR方法从 pc DNA- VEGF165质粒中扩增 VEGF165基因 ,在序列两端分别引入 Eco R 和 Bam H 限制性内切酶识别位点 ,并克隆到逆转录病毒表达载体 p LXSN质粒中。结果 :酶切鉴定、PCR扩增以及DNA序列分析表明已经将 VEGF165基因全长序列克隆到逆转录病毒表达载体 p LXSN质粒中 ,获得了 p LXSN- VEGF165重组质粒。结论 :成功地构建了人 VEGF c DNA逆转录病毒表达质粒 ,为进一步临床应用 VEGF c展开更多
文摘This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene, and the T-DNA region (152 bp).
文摘目的:建立重组 hPK-5质粒 DNA 基因治疗制剂的质量标准和检测方法。方法:采用限制性酶切图谱分析鉴定质粒DNA 结构和 PCR 法鉴定插入的 hPK-5基因作鉴别试验;采用分光光度法测定质粒 DNA 含量和 A_(260)/A_(280)比值;采用琼脂糖凝胶电泳法分析质粒 DNA 的超螺旋构象比例和残留 RNA 含量;采用阴离子交换色谱法分析 HPLC 纯度;采用 ELISA 法测定hPK～5蛋白的表达量;采用内皮细胞迁移法测定 hPK-5蛋白的生物学活性。结果:重组质粒 DNA 的限制性酶切片段大小与理论值一致;插入基因 PCR 扩增片段大小与理论值相符;质粒 DNA 含量为1.12 mg·mL^(-1);A_(260)/A_(280)比值为1.83;琼脂糖凝胶电泳法测定超螺旋构象质粒的比例为95.4%;HPLC 测定超螺旋质粒 DNA 占93.8%,开环质粒 DNA 占6.2%,琼脂糖凝胶电泳法测定木检出 RNA 残留;没有检出其他杂质;以2μg重组 hPK-5质粒转染5×10~5Hela 细胞48 h 后,2 mL 培养上清中 hPK-5蛋白浓度为109 ng·mL^(-1);以培养上清进行内皮细胞迁移试验,转染重组 hPK-5质粒 DNA 组迁移的内皮细胞数明显少于对照绀(P<0.05)。其他项目均符合相应的规定。结论:建立了重组 hPK-5质粒 DNA 基因治疗制剂的检定方法及其质量标准,为有效控制该类产品的质量打下基础。
文摘目的 :构建人血管内皮生长因子 ( VEGF) c DNA逆转录病毒表达质粒。方法 :采用 PCR方法从 pc DNA- VEGF165质粒中扩增 VEGF165基因 ,在序列两端分别引入 Eco R 和 Bam H 限制性内切酶识别位点 ,并克隆到逆转录病毒表达载体 p LXSN质粒中。结果 :酶切鉴定、PCR扩增以及DNA序列分析表明已经将 VEGF165基因全长序列克隆到逆转录病毒表达载体 p LXSN质粒中 ,获得了 p LXSN- VEGF165重组质粒。结论 :成功地构建了人 VEGF c DNA逆转录病毒表达质粒 ,为进一步临床应用 VEGF c
