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Adeno-associated virus mediated apoa-I and apoa-imilano expression in skeletal muscular cells

Journal of Nanjing Medical University 2005年第5期19卷 236-240页

作者： Ming Gui Zhiguang Wang Li Jiang Leming Fan Kejiang Cao Qi Chen Jun Huang Department of Cardiovascular Medicine the First Affiliated Hospital of Nanjing Medical University Nanjing 210029 Atherosclerosis Research Center Nanjing Medical University Nanjing 210029 China

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.

关键词： atherosclerosis apoa-I apoa-imilano adeno-associated viruse gene transfer

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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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Frontiers in Biology 2006年第4期1卷 345-348页

作者： ZHUANG Yingping MA Wenfeng GUO Meijin DING Mansheng CHU Ju ZHANG Siliang State Key Laboratory of Bioreactor Engineering National Research Center for BiotechnologyEast China University of Science and TechnologyShanghai 200237China State Key Laboratory of Bioreactor Engineering and National Research Center for Biotechnology East China University of Science and TechnologyShanghai 200237China

The effect of temperature on the formation of recombinant protein,apolipoprotein A-imilano was investigated in the present *** temperature of the initial growth phase was set at 30ºC,while temperature variation in induction phase was arranged in three *** cell-density culture of Escherichia coli and high expression of recombinant human by twice temperature-shifted induction were carried *** results showed that apoa-imilano reached 4.8 g/L with the final cell density of OD600,*** was found that twice temperature-shifted induction could successfully avoid the effect of acetic acid on cell density and the expression of the *** present study provides a basic procedure for the production of recombinant apoa-imilano.

关键词： recombinant Escherichia coli apoa-imilano high density high expression twice temperature-shifted induction

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