Vector of osteoporosis vaccine: expression, identification and purification of Qβ virus-like particles
ZHANG Shudong1, ZHOU Jian1, TIAN Zhuang1, LIU Changzhen2, YAO Qi1
1.Department of Orthopedics, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China; 2 Beijing Key Laboratory of Research of Chinese Medicine on Prevention and Treatment for Major Diseases, Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing 100700, China
Abstract：Objective To optimize expression and purification methods and prepare Qβ-VLPs with correct capsids structure and high purity and productivity.Methods We deleted the sequence of A1 gene sequence after termination codon of Qβ coat protein , and synthesized the gene sequence of Qβ coat protein only. The Qβ coat protein gene sequence was cloned into the pET30a vector plasmid, and the proteins were expressed by BL21(DE3), BL21(DE3) pLysS and Rosetta(DE3) strains. Then, the structure of Qβ-VLPs was verified with SDS-PAGE and transmission electron microscopy. Two different IPTG concentrations and three different methods of bacteria crushing were set respectively to optimize the expression conditions of the protein and methods of bacteria crushing. Finally, the supernatant of Qβ-VLPs was purified by ammonium sulfate precipitation, high-speed centrifugation and gel filtration chromatography.Results The results suggested that 5-6 polymers in SDS-PAGE and correct 25-30 nm capsids under electron microscopy were displayed only through the BL21(DE3)pLysS strain. The production of Qβ-VLPs expressed by IPTG at the concentration of 0.5mM was 2.8 times higher than at the concentration of 0.2mM, and the highest protein separation efficiency could be obtained by ultrasonic crushing of bacteria. The supernatant of Qβ-VLPs was purified and the purity of Qβ-VLPs was about 90%.Conclusions The experiment has explored the preparation strategy of Qβ-VLPs with correct capsids and high purity, which can contribute to the preparation and research of relevant vaccines.
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2019, Vol. 30 
