Abstract: 【Objective】The aim of this study was to clarify the subcellular localization of chicken ANP32 family member proteins and their expression characteristics in immune organs of chicken at different months,which laid a foundation for the follow-up study of the relationship between chicken ANP32 family member proteins and Newcastle disease virus(NDV) replication and pathogenicity.【Method】The recombinant eukaryotic expression vectors of chicken ANP32 family member genes were constructed,induced their expression after transfection into HEK-293T cells.The expression and subcellular localization of the above fusion proteins was examined by Western blotting and fluorescence observation,respectively. At the same time,the expression levels of chicken ANP32 family member genes in immune organs(spleen,thymus and bursa of Fabricius) at 1-to 6-month-old chicken was detected by real-time fluorescence quantitative PCR(RT-qPCR).【Result】The recombinant eukaryotic expression vectors(including pEGFP-C1-ANP32A,pEGFP-C1-ANP32B and pEGFP-C1-ANP32E) of chicken ANP32 family member genes were successfully constructed and the fusion proteins with EGFP tags EGFP-ANP32A,EGFP-ANP32B and EGFP-ANP32E were obtained after transfection into HEK-293T cells.Their molecular weights were 60.0,57.9 and 56.9 kD respectively. The results of subcellular localization analysis showed that the fluorescence of fusion proteins EGFP-ANP32A,EGFP-ANP32B and EGFP-ANP32E completely coincided with the nuclear fluorescence,that was,they were mainly located in the nucleus. The results of RT-qPCR assay showed that ANP32 family member genes were expressed in immune organs(spleen,thymus and bursa of Fabricius) of 1-to 6-monthold chicken,and the expression level of ANP32 family member genes was different in different immune organs. The expression of ANP32A and ANP32E genes in the immune organs of 1-to 6-month-old chicken increased first and then decreased,which reached the peak in the immune organs of 2-month-old chicken. The expression of ANP32B gene showed a downward trend,with the highest relative expression in the immune organs of 1-month-old chicken.【Conclusion】Chicken ANP32 family member proteins are mainly located in the nucleus,and ANP32 family member genes are expressed in the immune organs at different months,but the three genes have their own unique expression characteristics. Therefore,the relationship between different expression levels and the development of immune organs and immune function deserved to be further explored.