Screening Of The Bacteria Induced Genes And Analysis Of Their Promoter’s Activity In Goat Mammary Epithelial Cells

Goat mastitis is a very common disease which is a inflammation caused by bacterium orfungi infecting breast tissue. Mastitis not only decrease milk yield and quality, but also hurtgoat health. In order to detect the higher expressed immune factors during mastitis process, theexperiment was conducted as following: heat-inactivated Staphylococcus aureus, Streptococcusdysgalactiae and E.coli were used to stimulate goat mammary epithelial cells (GMECs) for0,2,4,6and8h, then LF, TNF-α, IL-1α, IL-1β, IL-8, Toll-2, Toll-4and BD-3mRNA was detectedrespectively. The results showed that after E. coli stimulation for6h, LF, IL-1α, IL-1β andToll-2, expressed higher than the control group (P<0.05); After Staphylococcus aureusstimulation for6h, TNF-α, IL-1α, IL-1β, IL-8, Toll-2and Toll-4expressed higher than thecontrol group (P<0.05); After dysgalactiae stimulation for6h, TNF-α, IL-1α, IL-1β and Toll-2expressed higher than the control group (P<0.05). When GMECs were stimulated byEscherichia coli, Staphylococcus aureus and Streptococcus dysgalactiae, IL-1α, IL-1β andToll-2expressed in a high level, which indicated that the three genes plays an important role inmastitis process. The result have lay the foundation for the mechanism research on mastitis.The development of a bacteria-inducible expression system has more advantages thanpersistent expression of anti-bacterial proteins in milk to combat mastitis. The present studywas conducted to determine whether those mastitis responsive promoters could regulate EGFPexpression in GMECs in response to E. coli, S. aureus or S. agalactiae infection. Theexpression level of IL-1α was significantly increased in E. coli, S. aureus and S. agalactiaeinfected GMECs than uninfected GMECs. The expression level of IL-1β was induced by E.coli and S. aureus, TLR2was induced by E. coli only. The promoters of IL-1α, IL-1β andTLR2were used to construct promoter-EGFP reporter gene vectors. The vectors were used totransfect GMECs. The expression level of EGFP was tested by flow cytometry analysis andwestern blot in transfected GMECs after bacterial infection. The expression of EGFP drove byIL-1α and IL-1β promoters were higher in E. coli, S. aureus, and S. agalactiae infected GMECsthan that of uninfected GMECs (P<0.05). The expression of EGFP drove by TLR2promoterwas not different between S. aureus, S. agalactiae infected GMECs and uninfected GMECs, butsignificantly increased when E. coli infection. These results indicated that the promoters of some bacterial-inducible genes could regulate EGFP expression in GMECs in response tobacterial challenges. Therefore, it was possible to control the expression of anti-bacterialprotein in mammary gland when bacteria invasion by this inducible expression strategy.β-defensin-3(BD-3) plays a crucial role in mastitis but at present little has been known inSaanen Dairy Goat. Based on published bovine BD-3coding sequence (CDS), a210-bpfragment from Saanen Dairy Goat was obtained by RT-PCR. It was identified as the CDS ofBD-3gene and submitted to Genbank, accession number is KJ433557. Through analyzing itsnucleotide and amino acid sequence between Saanen Dairy Goat and other species, we foundthat nucleotide sequence similarity was91.2%compareed with bovine, and80.9%with human.In the functional domains of the amino acid sequence at No.23-No.67, homology and similartertiary structure was99%compared with human. The result have lay the foundation for themechanism research on mastitis.