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Adeno-associated virus mediated apoA-I and apoA-IMilano expression in skeletal muscular cells
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作者 Ming Gui Zhiguang Wang +4 位作者 Li Jiang Leming Fan Kejiang Cao Qi Chen Jun Huang 《Journal of Nanjing Medical University》 2005年第5期236-240,共5页
Objective: To construct recombinant adeno-associated virus type 2(rAAV2) vectors encoding human apoA-I/apoA-lMilano protein, and explore an effective and safe method to prevent and treat the atherosclerotic disease... Objective: To construct recombinant adeno-associated virus type 2(rAAV2) vectors encoding human apoA-I/apoA-lMilano protein, and explore an effective and safe method to prevent and treat the atherosclerotic diseases. Methods: Human apoA-I cDNA with a His-tag in the upward stream of cDNA sequence were obtained with RT-PCR and PCR, and human apoA-IMilano cDNA was prepared by QuikChange Site-Directed Mutagenesis Kit. After extracted rAAV vectors with a most economic and convenient method, the particle numbers of rAAV vectors were measured by Dot-blot, and the purity was assayed by SOS-Page. The expression efficiency of the apoA-I or apoA-IMilano in C2C12 infected by rAAV vectors were detected by ELISA method. Results: ApoA-I cDNA was gained by RT-PCR and a His-tag was added in the upward stream of apoA-I cDNA successfully. ApoA-I cDNA was mutanted to apoA-IMilano cDNA successfully by QuikChange Site-Directed Mutagenesis Kit. The both titres of the rAAV vectors of apoA-I and apoA-IMilano were about 2×10^14/L, and the result of SOS-Page showed that the purity of the rAAV vectors was satisfied. The expression level of apoA-I was (0.39±0.04) μg/ml and the apoA-IMilano was (0.31±0.03) μ/ml in the DMEM culture medium at the first 24h after transfection. Conclusion: The success of the rAAV vectors construction, purification and the expression of apoA-I and apoA-IMilano in C2C12 cells mediated by these vectors, makes possible to inject rAAVA and rAAVAM vectors into mice muscle, and rises a new hope on finding a new way to prevent and treat atherosclerotic diseases and cardiovascular disease. 展开更多
关键词 ATHEROSCLEROSIS APOA-I apoa-imilano adeno-associated viruse gene transfer
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两次诱导实现重组大肠杆菌高密度、高表达研究 被引量:2
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作者 马文峰 庄英萍 +3 位作者 郭美锦 丁满生 储炬 张嗣良 《微生物学通报》 CAS CSCD 北大核心 2005年第2期54-59,共6页
通过不同温度诱导模式对大肠杆菌高密度,高表达的研究,确定两次升温诱导模式实现大肠杆菌高密度、高表达重组人载脂蛋白的目的。实验证实两次诱导成功的避免了乙酸对高密度、高表达的影响,最终发酵的细胞密度OD600达150,蛋白表达量4.8g&... 通过不同温度诱导模式对大肠杆菌高密度,高表达的研究,确定两次升温诱导模式实现大肠杆菌高密度、高表达重组人载脂蛋白的目的。实验证实两次诱导成功的避免了乙酸对高密度、高表达的影响,最终发酵的细胞密度OD600达150,蛋白表达量4.8g·L-1,证明两次升温诱导的发酵方法在高密度、高表达外源蛋白上是成功的,从而为基因工程菌规模化生产奠定了基础。 展开更多
关键词 重组大肠杆菌 ApoA—IMilano 高密度 高表达 两次诱导
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High cell density and high expression of recombinant human ApoA-I_(Milano) in Escherichia coli by twice temperature-shifted induction
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作者 ZHUANG Yingping MA Wenfeng +3 位作者 GUO Meijin DING Mansheng CHU Ju ZHANG Siliang 《Frontiers in Biology》 CSCD 2006年第4期345-348,共4页
The effect of temperature on the formation of recombinant protein,apolipoprotein A-IMilano was investigated in the present study.The temperature of the initial growth phase was set at 30ºC,while temperature varia... The effect of temperature on the formation of recombinant protein,apolipoprotein A-IMilano was investigated in the present study.The temperature of the initial growth phase was set at 30ºC,while temperature variation in induction phase was arranged in three modes.High cell-density culture of Escherichia coli and high expression of recombinant human by twice temperature-shifted induction were carried out.Experimental results showed that ApoA-IMilano reached 4.8 g/L with the final cell density of OD600,150.It was found that twice temperature-shifted induction could successfully avoid the effect of acetic acid on cell density and the expression of the product.The present study provides a basic procedure for the production of recombinant ApoA-IMilano. 展开更多
关键词 recombinant Escherichia coli apoa-imilano high density high expression twice temperature-shifted induction
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