TPO内含子Ⅴ可显著增强人血小板生成素基因在乳腺细胞中的表达

Mammary Gland-specific Expression Vectors with Intron Ⅴ of the Human Thrombopoietin Gene Facilitate Its High-level Expression in HC-11 cells

人血小板生成素(thrombopoietin,TPO)基因组包括6个外显子和5个内含子,内含子在其表达过程中可能扮演着重要作用.为了研究人TPO基因组中不同内含子对TPO表达的影响,构建可在转基因动物乳腺中高水平表达人TPO的乳腺特异性表达质粒.本研究以65kb的山羊β-casein启动子为调控元件,构建了包括人TPOcDNA(pTPOA)、TPOintronⅠ-TPOcDNA(pTPOB)、ΔTPOintronⅠ-TPOcDNA(pTPOC)、TPOintronⅤ-TPOcDNA(pTPOD)和TPOgDNA(pTPOE)等5种TPO乳腺特异性表达质粒,并在人乳腺细胞HC-11细胞上进行了瞬间表达研究.在转染48h后,通过双抗体夹心的ELISA方法定量分析上述质粒在HC-11细胞上的表达水平.结果表明,含有内含子Ⅴ的pTPOD的表达量最高,而含有整个基因组TPO的pTPOE表达水平最低.为了进一步证实pTPOD可在乳腺细胞中高水平表达,将pTPOD经脂质体包埋后注射到泌乳期山羊的乳腺中.结果显示,在山羊乳汁中可连续14d检测到人TPO的表达.上述实验证实,人TPO基因组中的内含子V可显著提高TPO在HC-11细胞内的表达水平,并提示内含子Ⅴ中可能含有特殊的调控序列.

Human thrombopoietin （TPO） stimulates the proliferation and maturation of megakaryocytes and regulates the production of platelets. The human TPO gene comprises six exons and five introns, which play different roles in TPO gene expression. To study the role of introns in expression of human TPO gene and obtain the mammary gland-specific expression vectors which can facilitate high-level expression of human TPO in mammary gland of transgenic animal, five mammary gland-specific expression vectors for TPO minigene were constructed with different exon-intron configurations fused to the regulatory elements of the 6.5 kb of goat β- casein gene. The TPO cDNA and gDNA were constructed by the same way to serve as controls. These recombinant vectors were introduced into cultured HC-11 cells for transient expression of TPO gene using lipofectin method. Forty-eight hours after transfection, the supernatant of cell culture was analysized using the human TPO immunoassay kit. The result showed that pTPOD, containing intron Ⅴ of the human TPO gene, induced the highest TPO expression level. To confirm this results, pTPOD mixed lipofectin was injected into the base of mammary gland of prolacting goats. Human TPO can be detected for two-weeks in the goat milk. The results showed that the intron Ⅴ of the human TPO gene enhances the TPO expression level in mammary gland cells, suggesting the presence of specific regulatory sequence in this intron.