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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (5): 40-47    DOI: 10.13523/j.cb.1912016
技术与方法     
易发生聚集的重组HBcAg病毒样颗粒的纯化*
谢航航1,2,白红妹1,2,叶超1,2,陈永俊1,2,袁明翠1,2,马雁冰1,2,**()
1 中国医学科学院北京协和医学院 医学生物学研究所 昆明 650118
2 云南省重大传染病疫苗研发重点实验室 昆明 650118
The Purification Procedure for the Recombinant HBcAg Virus-like Particle Easy to Generate Aggregation
XIE Hang-hang1,2,BAI Hong-mei1,2,YE Chao1,2,CHEN Yong-jun1,2,YUAN Ming-cui1,2,MA Yan-bing1,2,**()
1 Institute of Medical Biology,Chinese Academy of Medical Science&Peking Union Medical College,Kunming 650118, China
2 Yunnan Key Laboratory of Vaccine Research&Development on Severe Infectious Disease,Kunming 650118,China
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摘要：

目的:探索针对易发生聚集的重组HBcAg病毒样颗粒(VLP)的有效纯化方案。方法:培养的大肠杆菌经IPTG诱导重组HBcAg蛋白的表达,菌体超声破碎后的离心沉淀用含有不同浓度尿素的PBS缓冲液重悬溶解,经密度梯度离心并结合电镜观察对VLPs行为进行分析鉴定。以Sepharose 4 FF凝胶过滤层析在选定的尿素条件下纯化沉淀溶解液,纯化获得的目的蛋白进一步在含30%山梨醇的PBS中脱盐去除尿素。整个过程以SDS-PAGE及电镜进行各步骤样品中目的蛋白的分析。结果:含有1mol/L尿素的PBS缓冲溶液重悬超声沉淀,可有效溶解聚集的VLPs,在蔗糖密度梯度离心中显示典型HBcAg VLPs的行为,且电镜观察颗粒形态结构完整。经1mol/L尿素下凝胶过滤,VLPs进一步获得纯化。在脱尿素过程中流动相采用含30%山梨醇的PBS,有效避免了VLPs在尿素去除后重新聚集。结论:尿素与山梨醇的联合应用,为具有聚集现象的VLPs纯化制备提供了一种有效解决方案。
关键词： 病毒样颗粒;  纯化;  聚集;  尿素;  山梨醇    
Abstract:

Objective: To explore an effective purification procedure of recombinant HBcAg virus like particles (VLPs) that are prone to aggregation. Methods: The expression of recombinant HBcAg protein was induced by IPTG in cultured E. coli. The centrifugation precipitates of bacteria after ultrasonic fragmentation were resuspended and dissolved in PBS buffer with different concentrations of urea. The VLPs behavior was analyzed and identified by density gradient centrifugation and electron microscopy. The precipitate solution was purified by Sepharose 4 FF gel filtration chromatography under the selected urea conditions, and the purified target protein was further desalinate to remove urea with PBS containing 30% sorbitol. The entire process was analyzed by SDS-PAGE and electron microscopy. Results: The ultrasonic precipitates resuspensed with PBS buffer containing 1mol/L urea was effective dissolve the aggregated VLPs, which showed the behavior of typical HBcAg VLPs in sucrose density gradient centrifugation, and morphology and structure of the particales were complete by electron microscope. VLPs were further purified after 1mol/L urea gel filtration. In the process of urea removal, PBS containing 30% sorbitol was used as the mobile phase, which effectively avoided the reaggregation of VLPs after urea removal. Conclusion: The combined application of urea and sorbitol provides an effective solution for the purification and preparation of VLPs with aggregation phenomenon.
Key words: Virus-like particle (VLP)    Purification    Aggregation    Urea    Sorbitol
收稿日期: 2019-12-10 出版日期: 2020-06-02
ZTFLH:  Q939.4  
基金资助: * 国家自然科学基金面上项目(81773270);云南省应用基础研究重点项目(2016FA049)
通讯作者: 马雁冰     E-mail: may@imbcams.com.cn
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引用本文:

谢航航,白红妹,叶超,陈永俊,袁明翠,马雁冰. 易发生聚集的重组HBcAg病毒样颗粒的纯化*[J]. 中国生物工程杂志, 2020, 40(5): 40-47.

XIE Hang-hang,BAI Hong-mei,YE Chao,CHEN Yong-jun,YUAN Ming-cui,MA Yan-bing. The Purification Procedure for the Recombinant HBcAg Virus-like Particle Easy to Generate Aggregation. China Biotechnology, 2020, 40(5): 40-47.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1912016    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I5/40

图1  重组蛋白的表达鉴定
图2  以沉淀形式存在的重组蛋白在低浓度的尿素中以可溶性形式存在并为VLPs
溶解率和回收率 0.5mol/L 1mol/L 2mol/L 4mol/L
总沉淀(g) 0.162 0 0.162 0 0.162 0 0.162 0
剩余沉淀(g) 0.148 0 0.078 2 0.077 1 0.073 3
溶解沉淀(g) 0.014 0 0.083 8 0.084 9 0.088 7
沉淀溶解率(溶解沉淀/总沉淀×100%) 8.6% 51.7% 52.4% 54.8%
总蛋白质回收率[总蛋白质/(总蛋白质+沉淀蛋白质)×100%] 12.9% 80.2% 90.0% 92.1%
纯度(%) 41.5% 57.7% 55.9% 54.4%
目的蛋白回收率[总蛋白质×纯度/(总蛋白质+沉淀蛋白质) ×100%] 5.3% 46.3% 50.30% 50.10%
表1  不同浓度尿素溶解超声沉淀溶解率和蛋白质回收率
图3  凝胶过滤层析纯化重组蛋白
图4  不同保护剂溶解白色重组蛋白
重组蛋白溶解率 10%
山梨醇		 20%
山梨醇		 30%
山梨醇		 5%
海藻糖		 0.5mol/L
甘氨酸		 0.5mol/L
NaCl		 10%
甘油		 10%
蔗糖		 10%
葡萄糖
总沉淀(g) 0.031 0 0.031 0 0.031 0 0.031 0 0.031 0 0.031 0 0.031 0 0.031 0 0.031 0
剩余沉淀(g) 0.024 3 0.018 4 0.007 0.014 1 0.030 4 0.028 8 0.026 1 0.031 0 0.021 6
溶解沉淀(g) 0.005 7 0.012 6 0.023 0.015 9 0.000 6 0.001 2 0.004 9 0 0.008 4
溶解率(溶解沉淀/
总沉淀× 100%)		 18.4% 40.6% 74.2% 51.3% 1.9% 3.8% 15.8% 0% 27.1%
表2  不同保护剂溶解白色重组蛋白溶解率
图5  30%山梨醇缓冲体系凝胶过滤脱尿素
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