双域融合改善了Bst DNA聚合酶的逆转录酶活性和抑制剂耐受性
Rong Xiang1, Guang-Yi Liu2, Yi Hou3
1School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China.
International journal of biological macromolecules
|June 20, 2024
概括
研究人员通过将BstDNA聚合酶与DNA结合和疏水性蛋白质融合,设计了一种新奇的仿真DNA聚合酶HpStBL. 这种增强的聚合酶显示出对非洲猪瘟病毒和SARS-CoV-2检测的卓越抑制剂耐受性和活性.
科学领域:
- 分子生物学分子生物学
- 生物技术是生物技术.
- 酶工程是什么? 酶工程是什么?
背景情况:
- Bst DNA聚合酶对于DNA/RNA放大至关重要.
- 它的效率通常受到生物样本中存在的抑制剂的限制.
- 提高抑制剂的耐受性和活性对于直接检测方法至关重要.
研究的目的:
- 为了提高Bst DNA聚合酶的性能.
- 开发一种具有改进DNA/RNA放大效率和抑制剂耐受性的仿真聚合酶.
- 评估工程聚合酶在直接检测病原体方面的潜力.
主要方法:
- 通过与Sto7d和Hp47蛋白质融合,构建了四种仿真BstDNA聚合酶.
- 测试抑制剂对各种物质的耐受性,如肝素,湿酸和生物矩阵.
- 评估对非洲猪瘟病毒和SARS-CoV-2检测的敏感性和逆转录酶活性.
主要成果:
- 一种仿真蛋白HpStBL表现出最高的抑制剂耐受性.
- 在存在氨酸,酸,SYBR Green I,全血,组织和便的情况下,HpStBL保持高活性.
- 对于非洲猪瘟病毒检测,HpStBL表现出高灵敏度,对于检测SARS-CoV-2RNA的反转录酶活性也非常出色.
结论:
- 功能域与Bst DNA聚合酶的融合是提高性能的有效策略.
- 作为一种强大的DNA聚合酶,HpStBL显示出有前途的潜力,用于直接检测非洲猪瘟病毒和SARS-CoV-2病毒.
- 工程HpStBL聚合酶为异热放大试验提供了改善的抑制剂耐受性和逆转录酶活性.
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