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| Construction of a HEK-293T Cell Line Stable Overexpression of the KLHL21 Gene and Its Effect on Vesicular stomatitis virus Proliferation |
| LIU Qi1,2, CHEN Chuang-Wei1,2, LIU Si-Han3, SHAO Wen-Hua1,2, HUANG Meng-Yao1,2, YU Fan-Rui3, ZHENG Hai-Xue1,2, ZHANG Wei1,2,*, YANG Fan1,2,* |
1 College of Veterinary Medicine, Lanzhou University/State Key Laboratory of Animal Disease Control and Prevention/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China;
2 Gansu Province Research Center for Basic Disciplines of Pathogen Biology, Lanzhou 730046, China;
3 First Clinical Medical College of Lanzhou University, Lanzhou 730000, China |
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Abstract Kelch-like protein 21 (KLHL21), a member of the Kelch gene family, plays critical roles in various physiological processes in mammals, including mediating protein-protein interactions, protein degradation, and signal transduction. In order to study the effect of KLHL21 on the replication of Vesicular stomatitis virus (VSV), in this study, a KLHL21-overexpressing HEK-293T cell line (HEK-293T/KLHL21) was established using a lentiviral packaging system, and the replication efficiency of VSV in the cells was compared with that in wild-type HEK-293T cells. Firstly, in HEK-293T cells, the auxiliary plasmids psPAX2, pMD2.G and the newly constructed recombinant plasmid pLV-puro-3×Flag -KLHL21 were co-transfected to produce Lentivirus. Then, it was used to infect HEK-293T cells, and the positive cells HEK-293T/KLHL21 were obtained through puromycin selection. Western blot was used to assess VSV protein levels, while indirect immunofluorescence was applied to visualize VSV-GFP expression. Additionally, viral titers were determined to evaluate viral replication. The results showed that the proliferation ability of VSV in the HEK-293T/KLHL21 cell line was significantly weakened compared with HEK-293T cells. The result of qPCR revealed that, compared with wild-type cells, the established cell line activated the typeⅠ interferon response after VSV infection, which consequently led to a significant upregulation of the expression levels of its downstream genes. These findings indicated that KLHL21 activated the typeⅠinterferon signaling pathway and significantly inhibited the proliferation of VSV. This study provides theoretical basis and material support for further research on the antiviral mechanism of KLHL21.
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Received: 26 May 2025
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Corresponding Authors:
*zw20082008@126.com; yangfan02@caas.cn
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