稳定表达鸡己糖激酶Ⅱ的HD11细胞系构建及其对J亚群禽白血病病毒复制的影响OA

This study aimed to construct a HD11 cell line stably expressing chicken hexokinase II(HK-2)using a lentiviral vector system,thereby providing a cellular model to investigate the role of HK-2 in chicken hyperglycemia and viral infection.First,the chicken HK-2 gene sequence was amplified by PCR and cloned into the lentiviral vector pLV3-CMV-MCS-Puro.Subsequently,this construct was co-transfected with helper plasmids into 293T cells to package the lentivirus,while an EGFP-expressing lentivirus was packaged as a control.HD11 cells were infected with the viruses,and after 72 hours,a stable cell line was established through puromycin selection.The function of HK-2 was preliminarily validated by measuring glucose consumption and glucose 6-phosphate(glucose 6-phosphate,G6P)production.The established stable cell line was then infected with avian leukosis virus subgroup J(ALV-J)to assess the expression levels of glucose metabolism-related proteins and viral replication efficiency.The results showed a significant increase in HK-2 expression levels in the HK-2 stably transfected HD11 cell line following selection.Functional assays confirmed that HK-2 overexpression promoted glucose consumption and G6P production.Viral infection experiments demonstrated that HK-2 stable overexpression significantly enhanced ALV-J replication in HD11 cells,accompanied by a concurrent increase in viral Env protein expression.Furthermore,in ALV-J-infected HK-2-HD11 cells,the expression of key glucose metabolism proteins,including HK-2,PKM2,and LDHA,was correspondingly increased.In conclusion,this study successfully constructed an HD11 cell line stably expressing the chicken HK-2 gene,providing preliminary evidence that HK-2 overexpression promotes ALV-J replication and enhances the expression of glucose metabolism-related proteins.This work lays a foundation for further in-depth research on the role of HK-2 in the interplay between chicken metabolic regulation and viral infection.