Taq DNA聚合酶的分子改造及其在探针法qPCR直扩体系中的应用

doi:10.12141/j.issn.1000-565X.230090

华南理工大学学报(自然科学版) ›› 2024, Vol. 52 ›› Issue (4): 8-16.doi: 10.12141/j.issn.1000-565X.230090

Taq DNA聚合酶的分子改造及其在探针法qPCR直扩体系中的应用

胡松青¹ 袁家惠¹ 刘光毅¹^,² 侯轶³

^1.华南理工大学食品科学与工程学院, 广东广州 510640
^2.广州英赞生物科技有限公司, 广东广州 510555
^3.华南理工大学轻工科学与工程学院, 广东广州 510640

收稿日期:2023-03-06 出版日期:2024-04-25 发布日期:2023-07-12
作者简介:胡松青（1972-），男，教授，博士生导师，主要从事食品蛋白质（酶）结构生物学、天然产物活性物质分离与功能研究。E-mail:fesqhu@scut.edu.cn
基金资助:
广东省基础与应用基础研究基金资助项目(2021A1515220141)

Molecular Modification of Taq DNA Polymerase and Its Application in Probe-Based qPCR Direct Amplification System

HU Songqing¹ YUAN Jiahui¹ LIU Guangyi¹^,² HOU Yi³

^1.School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China
^2.Guangzhou Enzyvalley Biotech Co. , Ltd. , Guangzhou 510555, Guangdong, China
^3.School of Light Industry Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China

Received:2023-03-06 Online:2024-04-25 Published:2023-07-12
About author:胡松青（1972-），男，教授，博士生导师，主要从事食品蛋白质（酶）结构生物学、天然产物活性物质分离与功能研究。E-mail:fesqhu@scut.edu.cn
Supported by:
the Basic and Applied Basic Research Foundation of Guangdong Province(2021A1515220141)

摘要/Abstract

摘要：

Taq DNA聚合酶作为实时荧光定量聚合酶链式反应（qPCR）技术的核心组分，其性能优劣直接影响qPCR技术的进一步发展。然而，野生型Taq DNA聚合酶的耐抑制剂性能差、延伸性能不足。为获得具有高性能的Taq DNA聚合酶，采用基因工程技术将双链DNA结合蛋白Sso7d或Sto7d融合在野生型Taq DNA聚合酶的N端或C端，构建了4个均可溶表达的改造体，再经过耐受性测试筛选较优的改造体，结果显示：改造体Taq-Sto的耐受性最高，其热稳定性不受影响，且在1 s/kbp的延伸条件下能成功扩增靶标，表明Taq-Sto具有增强的延伸性能，在TaqMan探针法qPCR体系中对腐殖酸、单宁酸、全血等抑制剂同样表现出良好的耐受性。EMSA实验发现：Taq-Sto对DNA模板的结合亲和力有所提高，有利于增强Taq-Sto对模板的竞争力；将Taq-Sto应用于非洲猪瘟病毒（ASFV）的TaqMan探针法qPCR检测，与商品化试剂相比，Taq-Sto具有更低的ASFV检出限，且在体积分数为2%~6%的猪粪便样本或猪肉样本中的检测灵敏度分别为100.0%和85.4%，说明Taq-Sto在直扩qPCR检测领域更具有优势。

关键词: Taq DNA聚合酶, 双链DNA结合蛋白, 耐受性, 聚合酶链式反应

Abstract:

As the key component of quantitative real-time polymerase chain reaction （qPCR） technology, Taq DNA polymerase’s performance directly affects the further development of qPCR technology. However, the wild-type Taq DNA polymerase has inadequate properties in inhibitor tolerance and elongation performance. To obtain Taq DNA polymerase with high performance, this study fused the double-stranded DNA-binding protein Sso7d or Sto7d to the N-terminal or C-terminal of wild-type Taq DNA polymerase by genetic engineering technology, which four soluble expression transformants were constructed, and then the better transformant was screened by tolerance test. The results show that the better transformant Taq-Sto has the highest tolerance, with no impact on its thermal stability, and the target can be successfully amplified by Taq-Sto under the extension condition of 1 s/kbp, indicating that Taq-Sto has enhanced extension performance. It also shows good tolerance to humic acid, tannic acid and whole blood in TaqMan qPCR system. EMSA experiment shows that the binding affinity of Taq-Sto to DNA template is improved, which is beneficial to enhancing the competitiveness of Taq-Sto to DNA template. Taq-Sto was applied to the TaqMan qPCR detection of African swine fever virus (ASFV). Compared with commercial reagents, Taq-Sto has lower detection limit of ASFV, and the detection sensitivity in 2%~6% (volume fraction) pig fecal samples or pork samples is 100.0% and 85.4%, respectively, indicating that Taq-Sto has more advantages in the field of direct qPCR detection. The results provide a reference for the development of DNA polymerase with better performance, which is conducive to further promoting the practical application of qPCR technology.

Key words: Taq DNA polymerase, double-stranded DNA-binding protein, tolerance, polymerase chain reaction

中图分类号:

Q814

胡松青, 袁家惠, 刘光毅, 等. Taq DNA聚合酶的分子改造及其在探针法qPCR直扩体系中的应用[J]. 华南理工大学学报(自然科学版), 2024, 52(4): 8-16.

HU Songqing, YUAN Jiahui, LIU Guangyi, et al. Molecular Modification of Taq DNA Polymerase and Its Application in Probe-Based qPCR Direct Amplification System[J]. Journal of South China University of Technology(Natural Science Edition), 2024, 52(4): 8-16.

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模板质量浓度/（pg·μL^-1）	C_t
模板质量浓度/（pg·μL^-1）	Taq-Sto	商品试剂
112.00	29.04±0.06	28.83±0.13
28.00	31.45±0.12	31.40±0.15
7.00	33.52±0.20	34.50±0.01
1.80	35.86±0.90	36.53±0.69
0.44	36.41±0.03	39.14±0.32
0.11	38.71±0.52	40.28
阴性

猪粪样本添加量/%	C_t
猪粪样本添加量/%	Taq-Sto	商品试剂
0	28.89±0.01	28.24±0.04
2	30.36±0.10	28.48±0.02
4	29.86±0.22	28.91±0.14
6	29.93±0.22	31.14±0.24
8	29.69±0.18	38.40±0.88
10	30.62±0.32
12	30.94±0.45
14	33.62±0.37

猪肉样本添加量/%	C_t
猪肉样本添加量/%	Taq-Sto	商品试剂
0	28.36±0.03	28.49±0.10
5	28.77±0.05
10	29.23±0.10
15	30.25±0.14
20	31.29±0.15
25	32.90±0.26
30	36.51±0.37

Taq DNA聚合酶的分子改造及其在探针法qPCR直扩体系中的应用

Molecular Modification of Taq DNA Polymerase and Its Application in Probe-Based qPCR Direct Amplification System

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