Construction And Activity Detection On Bacteria Induced Promoter Of Goat Mammary Epithelial Cells

Goat mastitis, one of the most common goat diseases, is an inflammation resulting from the infection by bacterium or fungi. It can not only have a bad effect on goat health situation,but also decrease milk quality. IL-1(Interleukin-1) is a cytokine and functions on inflammation regulation. As an important element of gene expression vector, IL-1 promoter plays as a significant cis-element in IL-1 gene expression regulation. Nowadays, efficient and specific promoter is the key element on transgenic area. Bce1(β-casein element 1), a 160 bp sequence, has response elements for prolactin-dependent regulation and can attend prolactin-mediated regulation. These study results remind us that Bce1 may function through prolactin-mediated regulation and increase the promoter dependence on epithelial cells,leading to the influence on promoter tissue-specific expression function. In this study, we used dairy goat as a model. Bce1 placed in upstream of a truncated IL-1α promoter reconstitutes a promoter even more potent. Our study systematically studied Bce1 regulation on IL-1αtissue-specific function, compared promoter’s expression on HEK-293 T cell, goat lung epithelial cell and goat mammary epithelial cell, and finally explored the promoter activity and tissue specificity. The results are as follows:(1) Using LPS to stimulate GMECs for 0. 2. 4. 6. 8. 10 hours. By using RT-qPCR quantitative test, we validated the IL-1α gene bacteria inducibility in primary cells at different time point. The expression level of the gene increased obviously at 6 h(P<0.05).(2) The promoter of IL-1α gene(1575bp) was cloned from goat dairy gene genome and the Bce1 sequence(160bp) was cloned from bovine genome with high fidelity polymerase chain reaction(PCR). The eukaryotic expression vectors pGL4.10-IL1α and pGL4.10-IL1α-Bce1 were successfully constructed. Meanwhile, the promoter of IL-1α gene was proved to be active and bacterial inducible.(3) The function of eukaryotic expression vectors pGL4.10-IL1α and pGL4.10-IL1α-Bce1 were validated with dual luciferase report system. The results showed that Bce1’s function on IL-1α promoter appeared after LPS(Lipoteichoic acid from S. aureus) stimulation.More specifically: in GMEC group, the expression level of pGL4.10-ILα1-Bce1 stimulated by LPS increased significantly(P<0.05). Moreover, after LPS treatment, the expression level ofpGL4.10-ILα1-Bce1 in GMEC increased more than that in GLEC and 293T(P<0.05). So we can conclude that we have successfully constructed tissue-specific bacterial induced promoter.In this study we cloned IL-1α promoter sequence and bovine β-casein element1 and successfully constructed dual luciferase reporter vectors pGL4.10-IL1α and pG4.10-IL1α-Bce1 with pGL4.10. The bacteria inductivity of IL-1α promoter and the tissue specificity of Bce1-regulated IL-1α promoter were confirmed. This study has laid the theoretical and experimental basis for the improvement on clinical dairy goat mastitis prevention.