腺相关病毒5 蛋白质颗粒 由E.E.产生的 大肠杆菌无细胞蛋白质合成
Danielle Deuker1, Ernest Asilonu2, Daniel G Bracewell1
1Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London, WC1E 6BT, United Kingdom.
ACS synthetic biology
|August 23, 2024
概括
这项研究表明大肠杆菌无细胞蛋白质合成产生AAV5病毒样颗粒. 这种方法为生成用于基因疗法和疫苗的生物活性病毒载体提供了快速和低成本的替代方案.
科学领域:
- 生物技术是生物技术.
- 分子生物学分子生物学
- 病毒学 病毒学
背景情况:
- 重组腺相关病毒 (rAAVs) 对基因疗法和疫苗开发至关重要.
- 目前的rAAV制造是昂贵和缓慢的,需要使用替代生产方法.
研究的目的:
- 探索基于大肠杆菌的无细胞蛋白合成 (CFPS) 产生复合腺相关病毒血清型5 (AAV5) 病毒样颗粒 (VLPs) 的潜力.
主要方法:
- 在不同的温度下 (18°C和37°C) 使用基于大肠杆菌的CFPS构建表达的AAV5病毒蛋白3 (VP3).
- 使用N-终端的Strep-tag II用于VP3的亲和性净化.
- 使用动态光散射 (DLS) 和传输电子显微镜 (TEM) 进行了粒子特征分析.
- 在HeLa细胞中通过细胞内部化试验评估了VLP的生物活性.
主要成果:
- 较低的温度 (18°C) 显著增加了AAV5 VP3的溶解度,与37°C相比,产生了~90%的可溶性蛋白质.
- N-终端的Strep-tag II通过亲和染色学促进了VP3的有效隔离.
- DLS和TEM证实了大约20nm粒子的形成.
- 生产的AAV5 VLP表现出生物活性,成功地内化到HeLa细胞中.
结论:
- 基于大肠杆菌的CFPS是一种可行且创新的平台,用于快速合成AAV5VLPs.
- 这种方法产生了具有生物活性的AAV VLP,显示了基因治疗和疫苗应用的前景.
相关概念视频
Amyloid Fibrils
Amyloid fibrils are aggregates of misfolded proteins. Under most circumstances, misfolded proteins are either refolded by chaperone proteins or degraded by the proteasome. However, in the case of a mutation or a disease, these proteins can accumulate to form large clusters and often further assemble to form elongated fibers, called fibrils.
Amyloid deposits were observed as early as 1639 in the liver and the spleen. In 1854, Rudolph Virchow performed iodine staining, normally used to...
Amyloid deposits were observed as early as 1639 in the liver and the spleen. In 1854, Rudolph Virchow performed iodine staining, normally used to...
Amyloid Fibrils
Amyloid fibrils are aggregates of misfolded proteins. Under most circumstances, misfolded proteins are either refolded by chaperone proteins or degraded by the proteasome. However, in the case of a mutation or a disease, these proteins can accumulate to form large clusters and often further assemble to form elongated fibers, called fibrils.
Amyloid deposits were observed as early as 1639 in the liver and the spleen. In 1854, Rudolph Virchow performed iodine staining, normally used to...
Amyloid deposits were observed as early as 1639 in the liver and the spleen. In 1854, Rudolph Virchow performed iodine staining, normally used to...
Bacterial Gastroenteritis
Bacterial gastroenteritis, characterized by diarrhea, abdominal cramps, and vomiting, is often caused by ingestion of contaminated food or water and is frequently associated with pathogenic Escherichia coli strains. These microbes exploit two principal mechanisms to inflict disease.Shiga toxin–producing E. coli, also referred to as STEC—notably O157:H7—release Shiga toxins that target ribosomes, blocking protein synthesis. The B subunit of the toxin binds the host glycolipid receptor...
Hepatitis
Hepatitis is an inflammatory condition of the liver most commonly caused by hepatotropic viruses (A–E), though non-infectious causes such as alcohol and drugs also exist.Hepatitis AHepatitis A virus (HAV) is a non-enveloped RNA virus of the Picornaviridae family. It is primarily transmitted via the fecal-oral route, typically through ingestion of contaminated food or water. After ingestion, HAV enters the bloodstream through the oropharynx or intestinal epithelium and reaches the liver. The...
Amebiasis
Entamoeba histolytica, a protozoan parasite, is responsible for intestinal and extraintestinal amebiasis. Though a significant proportion of infections remain asymptomatic, approximately 50 million individuals annually are estimated to present with clinical disease, resulting in up to 100,000 deaths globally. The disease burden is disproportionately high in regions with lower socioeconomic status, such as parts of India, Africa, Mexico, and Latin America.Etiology and TransmissionThe infective...
Appendicitis
Appendicitis is an acute inflammatory condition of the vermiform appendix, most commonly caused by obstruction of its lumen. The appendix is a narrow, blind-ended pouch that extends from the cecum, making it particularly prone to obstruction. Causes include fecaliths, lymphoid hyperplasia (often after viral infections), parasites, tumors, or foreign bodies. This obstruction initiates a cascade of pathological changes.Luminal Obstruction and Early InflammationAfter obstruction, normal mucosal...


