Suppr超能文献

无内毒素的纯化方法从不溶性包涵体聚集体中分离牛病毒性腹泻病毒 E2 蛋白。

Endotoxin-free purification for the isolation of bovine viral diarrhoea virus E2 protein from insoluble inclusion body aggregates.

机构信息

Queensland Agricultural Biotechnology Facility, Agri-Science Queensland, Queensland, Australia.

出版信息

Microb Cell Fact. 2011 Jul 26;10:57. doi: 10.1186/1475-2859-10-57.

Abstract

BACKGROUND

Protein expression in Escherichia coli may result in the recombinant protein being expressed as insoluble inclusion bodies. In addition, proteins purified from E. coli contain endotoxins which need to be removed for in vivo applications. The structural protein, E2, from Bovine Viral Diarrhoea Virus (BVDV) is a major immunogenic determinant, and is an ideal candidate as a subunit vaccine. The E2 protein contains 17 cysteine residues creating difficulties in E. coli expression. In this report we outline a procedure for successfully producing soluble and endotoxin-free BVDV E2 protein from inclusion bodies (IB).

RESULTS

The expression of a truncated form of BVDV-E2 protein (E2-T1) in E. coli resulted in predominantly aggregated insoluble IB. Solubilisation of E2-T1 with high purity and stability from IB aggregates was achieved using a strong reducing buffer containing 100 mM Dithiothreitol. Refolding by dialysis into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630 resulted in a soluble but aggregated protein solution. The novel application of a two-phase extraction of inclusion body preparations with Triton X-114 reduced endotoxin in solubilised E2-T1 to levels suitable for in vivo use without affecting protein yields. Dynamic light scattering analyses showed 37.5% of the protein was monomeric, the remaining comprised of soluble aggregates. Mice immunised with E2-T1 developed a high titre antibody response by ELISA. Western hybridisation analysis showed E2-T1 was recognised by sera from immunised mice and also by several BVDV-E2 polyclonal and monoclonal antibodies.

CONCLUSION

We have developed a procedure using E. coli to produce soluble E2-T1 protein from IB, and due to their insoluble nature we utilised a novel approach using Triton X-114 to efficiently remove endotoxin. The resultant protein is immunogenic and detectable by BVDV-E2 specific antibodies indicating its usefulness for diagnostic applications and as a subunit vaccine. The optimised E. coli expression system for E2-T1 combined with methodologies for solubilisation, refolding and integrated endotoxin removal presented in this study should prove useful for other vaccine applications.

摘要

背景

大肠杆菌中的蛋白质表达可能导致重组蛋白以不溶性包涵体的形式表达。此外,从大肠杆菌中纯化的蛋白质含有内毒素,需要去除内毒素才能用于体内应用。牛病毒性腹泻病毒(BVDV)的结构蛋白 E2 是主要的免疫原决定簇,是亚单位疫苗的理想候选物。E2 蛋白含有 17 个半胱氨酸残基,这给大肠杆菌表达带来了困难。在本报告中,我们概述了一种从包涵体(IB)中成功生产可溶性和无内毒素 BVDV E2 蛋白的方法。

结果

在大肠杆菌中表达 BVDV-E2 蛋白的截断形式(E2-T1)主要导致聚合的不溶性 IB 聚集。使用含有 100mM 二硫苏糖醇的强还原缓冲液可从 IB 聚集体中以高纯度和稳定性溶解 E2-T1。通过透析到含有 0.2%Igepal CA630 的 50mM Tris(pH7.0)中进行复性,得到可溶性但聚集的蛋白质溶液。两相萃取包涵体制剂与 Triton X-114 的新应用可将溶解的 E2-T1 中的内毒素降低至适合体内使用的水平,而不影响蛋白质产量。动态光散射分析表明,37.5%的蛋白质为单体,其余为可溶性聚集体。用 E2-T1 免疫的小鼠通过 ELISA 产生了高滴度的抗体反应。Western 杂交分析表明,E2-T1 被免疫小鼠的血清以及几种 BVDV-E2 多克隆和单克隆抗体识别。

结论

我们已经开发了一种使用大肠杆菌从 IB 生产可溶性 E2-T1 蛋白的方法,由于其不溶性,我们利用 Triton X-114 的新方法有效地去除内毒素。所得蛋白具有免疫原性,可被 BVDV-E2 特异性抗体检测到,表明其在诊断应用和亚单位疫苗方面的有用性。本研究中提出的 E2-T1 的优化大肠杆菌表达系统以及用于溶解、复性和集成内毒素去除的方法,对于其他疫苗应用应该是有用的。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d617/3160874/7d48f0ec9d8b/1475-2859-10-57-1.jpg

相似文献

4
5
Truncated E2 of bovine viral diarrhea virus (BVDV) expressed in Drosophila melanogaster cells: a candidate antigen for a BVDV ELISA.
J Virol Methods. 2007 Sep;144(1-2):49-56. doi: 10.1016/j.jviromet.2007.03.023. Epub 2007 May 21.
8
Preparation of chicken IgY against recombinant E2 protein of bovine viral diarrhea virus (BVDV) and development of ELISA and ICA for BVDV detection.
Biosci Biotechnol Biochem. 2016 Dec;80(12):2467-2472. doi: 10.1080/09168451.2016.1217144. Epub 2016 Aug 2.
9
Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV) E2 protein.
PLoS One. 2017 Jun 5;12(6):e0178469. doi: 10.1371/journal.pone.0178469. eCollection 2017.
10
Sequence-optimised E2 constructs from BVDV-1b and BVDV-2 for DNA immunisation in cattle.
Vet Res. 2007 Nov-Dec;38(6):819-34. doi: 10.1051/vetres:2007037. Epub 2007 Aug 31.

引用本文的文献

1
Rapid Purification of Endotoxin-Free RTX Toxins.
Toxins (Basel). 2019 Jun 12;11(6):336. doi: 10.3390/toxins11060336.
2
Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV) E2 protein.
PLoS One. 2017 Jun 5;12(6):e0178469. doi: 10.1371/journal.pone.0178469. eCollection 2017.
3
Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins.
PLoS One. 2017 Mar 29;12(3):e0173778. doi: 10.1371/journal.pone.0173778. eCollection 2017.
4
Immunogenicity of Outer Membrane Proteins VirB9-1 and VirB9-2, a Novel Nanovaccine against Anaplasma marginale.
PLoS One. 2016 Apr 26;11(4):e0154295. doi: 10.1371/journal.pone.0154295. eCollection 2016.
5
Silica Vesicle Nanovaccine Formulations Stimulate Long-Term Immune Responses to the Bovine Viral Diarrhoea Virus E2 Protein.
PLoS One. 2015 Dec 2;10(12):e0143507. doi: 10.1371/journal.pone.0143507. eCollection 2015.
7
Removal of endotoxins from bacteriophage preparations by extraction with organic solvents.
PLoS One. 2015 Mar 26;10(3):e0122672. doi: 10.1371/journal.pone.0122672. eCollection 2015.

本文引用的文献

1
Inclusion bodies: a new concept.
Microb Cell Fact. 2010 Nov 1;9:80. doi: 10.1186/1475-2859-9-80.
2
Refolding solubilized inclusion body proteins.
Methods Enzymol. 2009;463:259-82. doi: 10.1016/S0076-6879(09)63017-2.
3
Selecting an appropriate method for expressing a recombinant protein.
Methods Enzymol. 2009;463:131-47. doi: 10.1016/S0076-6879(09)63011-1.
4
Production of recombinant proteins by microbes and higher organisms.
Biotechnol Adv. 2009 May-Jun;27(3):297-306. doi: 10.1016/j.biotechadv.2009.01.008. Epub 2009 Jan 31.
5
Evaluation of efficacy of mammalian and baculovirus expressed E2 subunit vaccine candidates to bovine viral diarrhoea virus.
Vaccine. 2009 Apr 14;27(17):2387-93. doi: 10.1016/j.vaccine.2009.02.010. Epub 2009 Feb 12.
7
Truncated E2 of bovine viral diarrhea virus (BVDV) expressed in Drosophila melanogaster cells: a candidate antigen for a BVDV ELISA.
J Virol Methods. 2007 Sep;144(1-2):49-56. doi: 10.1016/j.jviromet.2007.03.023. Epub 2007 May 21.
8
Differential expression of the Ebola virus GP(1,2) protein and its fragments in E. coli.
Protein Expr Purif. 2007 Jul;54(1):117-25. doi: 10.1016/j.pep.2007.02.004. Epub 2007 Feb 15.
10
Systemic immune responses in sheep, induced by a novel nano-bead adjuvant.
Vaccine. 2006 Feb 20;24(8):1124-31. doi: 10.1016/j.vaccine.2005.09.009. Epub 2005 Sep 19.

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验