山羊NFKBIA基因过表达载体构建与卵巢颗粒细胞增殖迁移效应研究OA

[Objective]The expression vector of NFKBIA gene was constructed by determining the bioinformatics characteristics and expression level of NFKBIA gene in the gonadal axis tissues of goats,and the regulatory effect of NFKBIA gene on proliferation and migration of ovarian granulosa cells(OGCs)was further analyzed.[Method]The physicochemical properties,phosphorylation sites,glycosylation sites,functional domains,interacting proteins,and secondary and tertiary spatial structures of the NFKBIA protein were predicted by using bioinformatics software.The expression of NFKBIA gene in the tissue of goat gonadal axis was detected by real-time fluorescent quantitative PCR technology.The effects of the overexpressed plasmid on proliferation and migration ability of OCGs were investigated by constructing a NFKBIA gene overexpression vector,cell transfection,CCK-8,and cell scratch assays.[Result]The NFKBIA protein was a highly unstable hydrophilic protein consisting of 314 amino acids with a pI value of 4.53,and its main structural form was random coil with 27 potential phosphorylation sites and without glycosylation sites.NFKBIA protein primarily functioned in the nucleus,cytoplasm,and cell membrane,and exhibited high homology with human and bovine counterparts.The NFKBIA gene was expressed in the cervix,corpus uteri,cornua uteri,fallopian tube,ovary,hypothalamus and pituitary gland.The relative expression level of the NFKBIA gene in the fallopian tubes of multiparous ewes was the highest,while the relative expression level in the cervix was the lowest.The relative expression level of the NFKBIA gene in the cervix and corpus uteri of the single-lamb group was significantly higher than that of the multiple-lamb group,but the relative expression level of the NFKBIA gene in the cornua uteri,fallopian tube,ovary and hypothalamus of the single-lamb group was significantly lower than that of the multiple-lamb group.The overexpression vector of NFKBIA gene was successfully constructed,and the transfected OGCs significantly promoted cell proliferation and migration.[Conclusion]The NFKBIA protein is a highly unstable hydrophilic protein with an irregular coiled structure,and plays a role in the cell nucleus,cytoplasm and cell membrane.The NFKBIA gene is expressed in the gonadal axis tissues of goats,with the highest relative expression in the oviducts of multiparous ewes and the lowest in the cervix.The overexpression of NFKBIA gene significantly promotes proliferation and migration of OGCs in the gonadal axis tissues.