1. 轮状病毒 NSP1 和 NSP3 蛋白真核表达载体构建、表达及其调控Ⅰ型干扰素信号通路的功能.
- Author
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李凤迪, 王嘉瑞, 刘新宇, 黄海岩, 叶丽萍, 曾艳, and 曹欣
- Subjects
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RECOMBINANT proteins , *GENETIC vectors , *REPORTER genes , *T cells , *PROTEIN expression , *TYPE I interferons - Abstract
[Objective] The present paper aimed to construct the eukaryotic expression vector of rotavirus(Rotavirus, RV)non-structural protein1(NSP1)and 3(NSP3)and express it in human embryonic kidney epithelial HEK239 T cells, and then to study the preliminary effect of rotavirus NSP3 on the activity of IFN-β/ISRE reporter gene in vitro. [Method] First, the NSP1 and NSP3 coding genes of ape rotavirus SA11 strain were synthesized and cloned into the cloning vector pUCm-T, the target fragments of NSP1 and NSP3 were amplified by PCR, and then the eukaryotic expression plasmids specifically expressing recombinant proteins pRK-Flag-NSP1/pRK-HA-NSP1 and pRK-Flag-NSP3/pRK-HA-NSP3 were constructed by using eukaryotic expression vectors pRK-Flag and pRK-HA as skeletons. The plasmids were transfected into HEK293 T cells after restriction endonuclease digestion and sequencing. The expression of NSP1/NSP3 protein was identified by Western blot. Then, the recombinant plasmid pRK-Flag-NSP1/pRK-Flag-NSP3 and IFN-β-Luc/ISRE-Luc plasmid were co-transfected into HEK239 T cells. After stimulated by RIG-IN(RIG-I activation domain), the effect of NSP1/NSP3 protein on the activity of IFN-β/ISRE reporter gene was determined by double luciferase reporter gene system. [Result] The full-length genes of NSP1 and NSP3 were cloned successfully, and the eukaryotic expression plasmids pRK-Flag-NSP1/pRK-HA-NSP1 and pRK-Flag-NSP3/pRK-HA-NSP3, of NSP1 and NSP3 protein of ape rotavirus strain SA11 were constructed. The successful expression of NSP1 and NSP3 protein was confirmed by transfection and expression of Western blot in HEK293 T cells. Recombinant plasmids pRK-Flag-NSP1 and pRK-Flag-NSP3 could significantly reduce the activity of double luciferase induced by RIG-IN. [Conclusion] NSP1 and NSP3 proteins can inhibit the activities of IFN-β and ISRE promoters stimulated by RIG-IN, which proves that NSP3 protein has the same function as NSP1 protein in inhibiting IFN-β signal pathway, which lays a theoretical foundation for further study on the mechanism of NSP3 protein regulating type I interferon signal pathway. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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