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| Establishment of Oryctolagus cuniculus RK13 Cell Line Stably Expressing ANXA2 and Study on Its Adsorption for RHDV |
| LONG Cui-Qin, WU Xiao-Chun, YUE Ya-Hui, ZHANG Hong-Yan, ZHANG Li, MA Hai-Yun, XING Xiao-Yong, WEN Feng-Qin, BAO Shi-Jun* |
| College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China |
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Abstract Rabbit hemorrhagic disease (RHD) is a highly lethal infectious disease of rabbits (Oryctolagus cuniculus) caused by Rabbit hemorrhagic disease virus (RHDV). Annexin A2 (ANXA2) is closely associated with the infection process of many viruses. In order to investigate the role of rabbit ANXA2 in the process of RHDV infection of host cells, in this study, rabbit ANXA2 gene fragments were amplified by PCR, and the eukaryotic expression vector pCD513B-ANXA2 was constructed and transfected into 293T cells with packaging plasmids pLP1, pLP2, and pLP/VSVG, then packaged lentiviral particles containing ANXA2 gene, infected rabbit kidney cells (RK13), and screened the cell line RK13-513B-ANXA2 for stable overexpression of ANXA2 protein using puromycin, detected the expression of ANXA2 gene and its protein by qPCR and Western blot. siRNA was synthesized and transfected into RK13 to analyze the gene expression. After 2 h, the relative change of RHDV adsorption was detected by qPCR, immunological fluorescence assay (IFA) and Western blot. The results showed that viral particles with high titers of 5.0×107~1.0×108 TU/mL were obtained using lentiviral vector packaging. The expression of ANXA2 gene and its protein in RK13-513B-ANXA2 cells was significantly up-regulated (P<0.01). The adsorption of RHDV by RK13 overexpressing ANXA2 protein was significantly up-regulated (P<0.01), and interference with ANXA2 expression protein significantly down-regulated the adsorption of RHDV to RK13 (P<0.05). The results suggest that ANXA2 may be involved in the infection of host cells by RHDV, which provides a firm basis for further investigation of the mechanism of RHDV infection in host cells.
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Received: 29 December 2021
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Corresponding Authors:
*bsjdy@126.com
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