1 School of Life Science and Engineering, Foshan University/Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, Foshan 528225, China;
2 Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Abstract African swine fever (ASF), a devastating disease for pigs (Sus scrofa), usually causes hemorrhagic fever, which bring great economic losses to the pig industry. Multigene families (MGF) 505-5R was in the right variable region (RVR) of the African swine fever virus (ASFV) genome and could influence the virulence of the virus. The work aimed to analyze the molecular characteristics of the MGF505-5R gene and protein and construct a eukaryotic expression vector for the ASFV MGF505-5R gene. The MGF505-5R gene was obtained from the ASFV reference genome in GenBank (GenBank No. MK128995.1) for bioinformatics analysis, and the MGF505-5R gene was synthesized. The MGF505-5R gene was ligated into the pRK5M-C-2×Strep vector by homologous recombination. The recombinant plasmid pRK5M-C-2×Strep-MGF505-5R was transfected into IPEC-J2 cells after being identified by PCR and sequencing, and the expression of the MGF505-5R gene was confirmed by immunofluorescence and Western blot. Transcript levels of exosome 8 subunits, Mtr4 exosome RNA helicase (MTREX), terminal nucleotidyltransferase 4A (TENT4A) and zinc finger DHHC-type palmitoyltransferase 16 (ZDHHC16) in IPEC-J2 cells overexpressing pRK5M-C-2×Strep-MGF505-5R were detected by qPCR. Bioinformatics analysis showed that the MGF505-5R gene was divided into 3 branches in the phylogenetic tree and there was a positive selection at amino acids 20, 105 and 485. The secondary structure of MGF505-5R protein matched the tertiary structure, and the positive selection sites were all located on the irregularly coiling. PCR results showed that the MGF505-5R gene fragment was successfully ligated into the pRK5M-C-2×Strep vector. Immunofluorescence and Western blot assays showed that the pRK5M-C-2×Strep-MGF505-5R vector was successfully expressed in porcine intestinal columnar epithelial cells (IPEC-J2) cells. The qPCR results showed that the expression of exocytotic complex subunits exosome component (EXOSC) 1, EXOSC2, EXOSC3, EXOSC4, EXOSC5, EXOSC7, EXOSC8, EXOSC10, TENT4A and ZDHHC16 was extremely significantly decreased in IPEC-J2 cells overexpressing pRK5M-C-2×Strep-MGF505-5R (P<0.01); no significant change in MTREX expression. The analysis of the molecular characteristics of MGF505-5R in this study provided a basis for the evolutionary study of this gene; the construction of the MGF505-5R expression vector in pig IPEC-J2 cells provides the effective tool and experimental material for the study of protein interactions between ASFV and the host.
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