非洲猪瘟病毒跨膜蛋白pX69R的表达及其亚细胞定位分析OA

The complete X69R gene of African swine fever virus(ASFV)was subcloned into the prokar-yotic expression vector pET-32a(+)by homologous recombination technology.The His-X69R recombinant protein was obtained through IPTG induction and nickel-affinity purification under denaturing condi-tions.The purified His-X69R protein was used to immunize 8-week-old mice to prepare anti-pX69R poly-clonal antibodies.The prepared polyclonal antibodies could specifically recognize FLAG-X69R recom-binant protein expressed in eukaryotic cells and pX69R protein expressed in ASFV-infected porcine alveolar macrophages(PAM).The molecular weight of pX69R protein band in ASFV-infected PAM cells was located at approximately 14 kDa and was primarily expressed in the cytoplasm.Bioinformatics analysis revealed that pX69R lacks a signal peptide,and its amino acids(aa)21-38 constitute a potential trans-membrane domain.Subcellular localization assay indicated that pX69R largely overlapped with endo-plasmic reticulum(ER)signal but showed no significant colocalization with the Golgi apparatus.Trun-cated mutants were further constructed based on the predicted structural domains such as transmembrane region.Subcellular localization analysis demonstrated that deletion of only the 21-38 aa transmem-brane region did not affect ER localization,whereas deletion of 1-41 aa altered its ER localization.These results lay a foundation for further research on the mechanism of pX69R in the replication and pathogenesis of ASFV.