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| Construction and Identification of a C129R Gene Double-tagged Recombinant African swine fever virus |
| GAN Lu-Lu, ZHOU Yan-Long, REN Jing-Jing, YAN Li-Jiao, ZHENG Hai-Xue*, LI Dan* |
| Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China |
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Abstract African swine fever (ASF) is a highly lethal infectious disease caused by African swine fever virus (ASFV), inflicting devastating losses on the global pig industry. Currently, no safe and effective commercial vaccines are available, and elucidating the functions of viral key proteins serves as the core prerequisite for vaccine development and antiviral strategy design. Traditional research on ASFV protein functions heavily relies on specific antibodies, which is limited by long preparation cycles and cumbersome procedures. C129R is a structural protein encoded by the central region of ASFV, while its precise localization in viral particles, dynamic distribution and host interaction mechanisms remain unclear. To investigate the functions of C129R during viral infection and pathogenesis, in this study, a recombinant ASFV strain with a C-terminal 3×Flag-Strep double tag fused to the structural protein C129R was constructed. Using homologous recombination technology, the gene sequences encoding the 3×Flag-Strep double tag and GFP selection marker were inserted into the C-terminus of the C129R gene of ASFV CN/GS/2018 strain, and the recombinant virus ASFV-C129R-3×Flag-Strep was obtained through clonal screening and purification. Western blot analysis confirmed that the recombinant virus correctly expressed the C129R fusion protein carrying the double tag. qPCR revealed that tag insertion had no significant effect on the transcription levels of C129R and its upstream and downstream genes M448R and C84L. One-step growth curve, hemadsorption assay and transmission electron microscopy showed no significant differences in in vitro replication kinetics, morphological structure and viral assembly between the recombinant virus and the parental strain.This study successfully constructed a double-tagged recombinant ASFV targeting C129R with stable biological characteristics, providing a technical reference for further analyzing the precise localization and structural features of C129R in viral particles, as well as its interaction mechanism with host proteins.
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Received: 03 February 2026
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Corresponding Authors:
*lidan@caas.cn; zhenghaixue@caas.cn
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