建立重组人睫状神经营养因子(recombinant human ciliary neurotrophic factor,rhCNTF)的肽图分析方法,用于rh-CNTF的质量控制。胰蛋白酶对rhCNTF进行酶切后,利用RP-HPLC方法对酶切液进行分析,以获得胰蛋白酶切最佳条件及色谱条件,并对...建立重组人睫状神经营养因子(recombinant human ciliary neurotrophic factor,rhCNTF)的肽图分析方法,用于rh-CNTF的质量控制。胰蛋白酶对rhCNTF进行酶切后,利用RP-HPLC方法对酶切液进行分析,以获得胰蛋白酶切最佳条件及色谱条件,并对连续3批样品进行分析。rhCNTF的胰蛋白酶最佳酶切条件为37℃酶切24h,以A(0.1%TFA-H2O)、B(0.1%TFA-CH3CN)为流动相,采用梯度洗脱的方法对酶切液进行分析,结果连续3批rhCNTF制备产品的肽图完全一致,且其检出峰数目与理论推测值相符。3批产品肽图的一致性为rhCNTF产品的结构同一性提供了有利证据,同时,建立了rhCNTF产品质量控制的一项指标。展开更多
The recombinant human ciliary neurotrophi factor(hCNTF)expressed in E.coli aggregatedas inclusion bodies and refolding procedure was necessary to obtine the active protein.To overcome the disadvantage,we cloned hcntf ...The recombinant human ciliary neurotrophi factor(hCNTF)expressed in E.coli aggregatedas inclusion bodies and refolding procedure was necessary to obtine the active protein.To overcome the disadvantage,we cloned hcntf gene into yeast expression plasmid pPIC9K and collected the plasmid pPIC9K-hcntf.Plasmid pPIC9K-hcntf was transformed into yeast Pichia pastoris GS115,and screened on G418-SD plates.The transformants with high copies of hcntf gene were inoculated into BMMY media and induced with 0.5% methanol.The recombinant hCNTF was secreted into the media.The amount of hCNTF in the supernatant was about 10 mg/L when incubated in the conical flasks and reached up to 60 mg/L under fed-batch condition in 15 L fermentator.The recombinant hCNTF expressed in E.coli was renatured as the control.The neonatal rat dorsal root ganglion assay showed that proteins expressed in both systems have the activity of promoting the growth of neuron axons.The phenomenon can be observed with only 3 μg hCNTF expressed in yeast present,which indicates that hCNTF was successfully expressed in Pichia pastoris and has a relatively high activity.展开更多
文摘建立重组人睫状神经营养因子(recombinant human ciliary neurotrophic factor,rhCNTF)的肽图分析方法,用于rh-CNTF的质量控制。胰蛋白酶对rhCNTF进行酶切后,利用RP-HPLC方法对酶切液进行分析,以获得胰蛋白酶切最佳条件及色谱条件,并对连续3批样品进行分析。rhCNTF的胰蛋白酶最佳酶切条件为37℃酶切24h,以A(0.1%TFA-H2O)、B(0.1%TFA-CH3CN)为流动相,采用梯度洗脱的方法对酶切液进行分析,结果连续3批rhCNTF制备产品的肽图完全一致,且其检出峰数目与理论推测值相符。3批产品肽图的一致性为rhCNTF产品的结构同一性提供了有利证据,同时,建立了rhCNTF产品质量控制的一项指标。
文摘The recombinant human ciliary neurotrophi factor(hCNTF)expressed in E.coli aggregatedas inclusion bodies and refolding procedure was necessary to obtine the active protein.To overcome the disadvantage,we cloned hcntf gene into yeast expression plasmid pPIC9K and collected the plasmid pPIC9K-hcntf.Plasmid pPIC9K-hcntf was transformed into yeast Pichia pastoris GS115,and screened on G418-SD plates.The transformants with high copies of hcntf gene were inoculated into BMMY media and induced with 0.5% methanol.The recombinant hCNTF was secreted into the media.The amount of hCNTF in the supernatant was about 10 mg/L when incubated in the conical flasks and reached up to 60 mg/L under fed-batch condition in 15 L fermentator.The recombinant hCNTF expressed in E.coli was renatured as the control.The neonatal rat dorsal root ganglion assay showed that proteins expressed in both systems have the activity of promoting the growth of neuron axons.The phenomenon can be observed with only 3 μg hCNTF expressed in yeast present,which indicates that hCNTF was successfully expressed in Pichia pastoris and has a relatively high activity.
