Expression, purification, and renaturation of a recombinant peptide-based HIV vaccine in Escherichia coli.
To design an epitope-based vaccine for Human immunodeficiency virus (HIV), we previously predicted 20 potential HIV epitopes using bioinformatics approaches. The combination of these 20 epitopes has a theoretical coverage of 98.1% of the population for both the prevalent HIV genotypes and Chinese human leukocyte antigen DR types. To test the immunogenicity of this vaccine in vivo, a corresponding antigen needs to be prepared. To this end, we constructed a recombinant plasmid containing DNA encoding the epitopes and GPGPG spacers and a 6-His tag for verification of protein expression and ease of purification, and then transformed Escherichia coli cells with the plasmid. After IPTG induction, the recombinant protein was expressed in the form of mainly inclusion bodies. To stabilize the structure of denatured inclusion bodies for efficient purification and renaturation in vitro, we transferred the dissolved inclusion bodies from 7 mol/L guanidine hydrochloride to 8 mol/L urea. Under denaturing conditions, the vaccine protein was purified by a 3-step process including ion-exchange chromatography and affinity column, and then renatured by stepwise dialysis. Together, the above described procedures generated 43 mg of vaccine protein per litre of fermentation medium, and the final product reached approximately 95% purity. The purified protein was capable of eliciting antigen-specific T-cell responses in immunized mice.
a State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
Wei
W
He
b Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China.|c Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology of Ministry of Education/Health, Fudan University, Shanghai 200030, People's Republic of China.
Jiayi
J
Shu
a State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
Jian
J
Zhang
b Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China.
Zhihua
Z
Liu
c Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology of Ministry of Education/Health, Fudan University, Shanghai 200030, People's Republic of China.
Jianqing
J
Xu
b Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China.
Xia
X
Jin
a State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
Xuedong
X
Wang
D016915:AIDS Vaccines / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D010957:Plasmids; D022223:Vaccines, Subunit / Q000235:genetics / Q000302:isolation & purification; D014614:Vaccines, Synthetic / Q000235:genetics / Q000302:isolation & purification
D016915:AIDS Vaccines; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic
corps d’inclusion; epitope-based HIV vaccine; expression procaryote; immunogenicity; immunogénicité; inclusion body; prokaryotic expression; purification; vaccin anti-VIH à base d’épitopes