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

Posted on:2017-01-01Degree:MasterType:Thesis
Country:ChinaCandidate:X Y LinFull Text:PDF
GTID:2283330485478657Subject:Clinical Veterinary Medicine
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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.
Keywords/Search Tags:Bovine β-casein element1, mammary gland tissue specificity, bacteria induced, goat mammary epithelial cell
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