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| Affinity Purification of Enterovirus 71 Fused Multi-Epitope Protein Antigen and Assembling It as Virus-like Particles in Vitro |
| XUE Ling1,2, LIU Jiang-ning3, ZHANG Yao1,2, ZHANG Chun1, WANG Qi1,2, QIN Chuan3, LIU Yong-dong1, SU Zhi-guo1 |
1 National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China;
2 University of Chinese Academy of Sciences, Beijing 100049, China;
3 Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing 100021, China |
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Abstract Human enterovirus 71 (EV71) is one of the main causative pathogens for hand-foot-and-mouth disease (HFMD) which often infects infants and children under 5 years old. Although prophylactic vaccines of inactivated EV71 virus for HFMD have been clinically available, low productivity and safety concerns still prompt scientists to develop other vaccine substitutes. Recently, a kind of vaccine candidate VacA for EV71 has been designed and reported, which contained the main antigen peptides, was fused as a single protein and expressed through recombinant E. coli system. This candidate was demonstrated to have high neutralizing antibody titers as well as efficiently protect against virus infection. However, forming inclusion bodies in E. coli and failure of being separated from the host contaminants through route chromatographs hampered its path to go forward to whole pre-clinic and safety assessments.Another form of VacA as His-VacA was constructed by inserting a His tag at the N-terminal of VacA and explored to be separated through metal affinity chromatography. As still forming inclusion bodies, the fusion protein of His-VacA was directly purified at denatured state through a special mental affinity medium of cOmplete His-tag, which could tolerate low concentration of EDTA and DTT. Purity of about 95% for His-VacA was achieved after such one step chromatographic procedure, with a recovery of 46.8%. Dialysis was then adopted to remove urea in the purified protein, but protein almost completely precipitated when dialyzing against PB buffer without any additives. Nevertheless, in the case that the purified denatured protein was firstly diluted in a buffer containing 2mol/L urea and then exchanged to a buffer without urea through a desalting column of G25, no protein precipitate could be found, resulting in nearly 100% recovery. More inspirationally, His-VacA was found in the form of virus-like particles with a size of 10nm through transmission electron microscopy. Moreover, this protein particle is very stable in the final buffer and then could be used for the further pre-clinic assessments. All these results lay a foundation for developing His-VacA as a new kind of EV71 vaccine with the advantages of safer and cheaper.
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Received: 03 February 2016
Published: 02 March 2016
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