Tao Lihan, Wu Chengcheng, Lin Cui, Kang Zhaofeng, Huang Jianzhen
College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, China.
Institute of Animal Husbandry and Veterinary Medicine, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, Jiangxi, China.
Sheng Wu Gong Cheng Xue Bao. 2024 Dec 25;40(12):4573-4585. doi: 10.13345/j.cjb.240039.
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 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 5' 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 Mgconcentrations 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.
猪德尔塔冠状病毒（PDCoV）是导致哺乳仔猪致命性腹泻的主要病原体，目前缺乏预防和控制该病毒的有效疫苗及药物。非结构蛋白13（NSP13）作为病毒编码的解旋酶，被认为是抗病毒药物的关键靶点，因此研究NSP13的解旋酶活性势在必行。在本研究中，合成了PDCoV的基因并将其整合到原核表达载体pET-28a中，构建重组质粒pET-28a-NSP13。NSP13在BL21（DE3）中成功表达，随后进行了纯化。该研究还验证了纯化后的NSP13的解旋酶活性，并探究了影响该活性的因素。结果表明，PDCoV的NSP13在原核系统中有效表达并表现出解旋酶活性，能够解开5'端带有尾巴的双链DNA。此外，NSP13通过促进单链核苷酸链的互补配对形成双链而表现出退火功能。NSP13的解旋酶活性受金属离子影响，但0.5-6.0 mmol/L范围内的Mg浓度对NSP13的解旋酶活性无显著影响。当溶液pH值在4-9范围内时，解旋酶活性无差异。0.25-6.00 mmol/L范围内的ATP浓度对解旋酶活性有微弱影响，且NSP13浓度≥80 nmol/L会抑制解旋酶活性。我们获得了PDCoV的NSP13并研究了其解旋酶活性。这些发现为进一步研究NSP13在PDCoV复制中的调控机制以及开发抗冠状病毒药物提供了理论基础。
