目的 优化表达和纯化方式,制备出具有正确空间结构且纯度和产量较高的骨质疏松疫苗载体Qβ病毒样颗粒(Qβvirus-like particles,Qβ-VLPs)。方法 在基因合成时删除A1基因序列中Qβ衣壳蛋白终止密码子之后的序列,只合成Qβ衣壳蛋白的基因序列。将Qβ衣壳蛋白基因序列克隆到pET30a载体质粒上,用BL21(DE3)、BL21(DE3)pLysS、Rosetta(DE3)三种不同菌株进行蛋白表达,然后用SDS-PAGE和透射电镜验证是否表达出Qβ-VLPs的正确结构。设置2个蛋白表达诱导剂IPTG( Isopropyl β-D-1-thiogalactopyranoside,异丙基-β-D-硫代半乳糖苷)浓度和3种菌体破碎方式,分别优化蛋白表达条件和菌体破碎方案,然后将得到的Qβ-VLPs上清液通过硫酸铵聚沉、高速离心和分子筛分选进行Qβ-VLPs的纯化。结果 仅BL21(DE3)pLysS菌株可以表达出SDS-PAGE中呈现5-6聚体、透射电镜下呈现25~30 nm球形结构的Qβ-VLPs。使用0.5 mM浓度的IPTG表达的Qβ-VLPs产量较0.2 mM时高2.8倍,使用超声破碎菌体的方式可以获得较高的蛋白分离效率。将Qβ-VLPs上清液通过本实验过程中的方案进行纯化后纯度可达90%左右。结论 该实验探索出具有正确空间结构、组成单一且纯度较高的Qβ-VLPs的制备方案,为相关疫苗制备和研究奠定基础。
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.
关键词
骨质疏松症 /
Qβ病毒样颗粒 /
原核表达 /
蛋白纯化
Key words
osteoporosis /
Qβ-VLPs /
expression /
protein purification
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