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
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
文摘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.
基金This work was funded by the National Re-search Project for High-Tech Development(No.2002AA217021)supported by a Grant-aid for the Special Project of State Key Science and Technology.
文摘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.