Application of the Cre-LoxP System to Construct the Deleted TK Gene of the Lumpy Skin Disease Virus
A TK-deficient Lumpy skin disease virus(LSDV)strain was constructed by homolo-gous recombination technology and the Cre-LoxP system platform to provide an alternative strain for developing a safe and efficient LSDV genetic engineering vaccine.Using the TK gene as the target gene,using the Cre-LoxP system platform and red fluorescent protein(RFP)as screening marker,the left and right homologous arms and RFP gene expression cassettes were amplified by overlapping PCR and fused,and then cloned into pUC19T vector to construct gene deletion transfer vector pUC19T-LSDV△TK-RFP.The transfer vector recombinant plasmid was trans-fected into Vero cells and infected with LSDV/CHA/FJ/2021 strain to construct LSDV-△TK-RFP recombinant virus.LSDV-TK-RFP was screened and purified by the plaque method and lim-ited dilution method.Then,using the Cre-LoxP system,RFP tags were cut off from the viral ge-nome in MDBK-Cre cells,and LSDV-TK strains were screened and purified by the plaque method and limited dilution method.PCR and sequencing identified the recombinant strain,and its one-step growth curve was determined.The recombinant strain(LSDV-△TK)with deletion of the TK gene was successfully constructed,and the replication ability of the recombinant strain was slightly lower than that of the wild-type strain.The LSDV strain with deletion of the TK gene constructed by the Cre-LoxP system has good genetic stability and replication growth characteris-tics,so it can be used as an alternative strain for the development of the LSDV genetic engineer-ing vaccine and expands the application range of the Cre-LoxP system.
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