猪德尔塔冠状病毒NSP13蛋白原核表达与解旋活性分析

Prokaryotic expression and helicase activity analysis of PDCoV NSP13

摘要:

猪德尔塔冠状病毒(porcine deltacoronavirus, PDCoV)是导致哺乳仔猪致命性腹泻的一种重要病原体，目前缺少有效防治该病毒的疫苗和药物。非结构蛋白13 (NSP13)是一种病毒编码的解旋酶，被认为是抗病毒药物的重要靶点，因此有必要对其解旋活性进行研究。本研究以PDCoV的NSP13基因为模板，将其插入到原核表达载体pET-28a中，构建重组质粒pET-28a-NSP13。NSP13蛋白在大肠杆菌BL21(DE3)中成功表达并进行了纯化。验证了NSP13蛋白的活性，并且探究了影响NSP13解旋酶活性的因素。结果表明，PDCoV的NSP13蛋白能够在原核系统中表达，纯化后的NSP13具有解旋活性，能够解开具有5ʹtailed的双链DNA。此外，NSP13还具有促进核酸单链的碱基互补配对形成双链的退火功能。金属离子的种类会影响NSP13的解旋活性，但在0.5–6.0 mmol/L范围内的Mg²⁺浓度对NSP13解旋活性无明显影响。当溶液pH在4–9范围内，NSP13解旋活性无明显差异。在0.25–6.00 mmol/L范围内的ATP浓度对NSP13解旋活性存在微弱的影响，NSP13浓度≥80 nmol/L则会抑制解旋活性。本研究获得了重组表达的PDCoV NSP13蛋白并对其解旋酶活性进行了探究，为深入理解NSP13调控PDCoV复制的机制和抗冠状病毒药物的研发奠定了理论基础。

Abstract:

Porcine deltacoronavirus (PDCoV) is a major pathogen causing fatal diarrhea in suckling piglets, and there is currently a lack of effective vaccines and drugs to prevent and control the virus. The nonstructural protein 13 (NSP13) serves as a virus-coded helicase and is considered to be a crucial target for antiviral drugs, making it imperative to investigate the helicase activity of NSP13. In this study, the NSP13 gene of PDCoV was synthesized and integrated into the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-NSP13. NSP13 was successfully expressed in BL21 (DE3) and subsequently purified. The study also verified the helicase activity of the purified NSP13 and explored the factors that influence this activity. The results indicated that NSP13 from PDCoV was effectively expressed in the prokaryotic system and exhibited helicase activity, capable of unwinding double-stranded DNA with a tail at the 5ʹ end. Additionally, NSP13 demonstrated an annealing function by promoting the complementary pairing of single-stranded nucleotide chains to form double strands. The helicase activity of NSP13 was affected by metal ions, but Mg²⁺concentrations in the range of 0.5–6.0 mmol/L had no significant effect on helicase activity of NSP13. When the solution pH was in the range of 4–9, there was no difference in helicase activity. ATP concentrations in the range of 0.25–6.00 mmol/L had a weak effect on helicase activity, and NSP13 concentration ≥80 nmol/L inhibited the helicase activity. We obtained the NSP13 of PDCoV and investigated its helicase activity. These findings provided a theoretical foundation for the further research on the regulatory mechanism of NSP13 in PDCoV replication and the development of anti-coronaviral drugs.