| MMP-9(Matrix metalloproteinase-9, Gelatinase B), is synthesis and secretion by varietytypes of cells. The main function of MMP-9is to degradate the compositions of extracellularmatrix and regulate the activity of the soluble proteins and involved in inflammation andtissue remodeling. Studies have shown that MMP-9activity was increased in milk of cowsand goats which were suffered with mastitis. But the function of MMP-9in the mechanismhow the mastitis happen and how to regulation of milk somatic cell counts in milk is not clear.So far, there is still no complete sequence of MMP-9gene was cloned in goats. In this study,we cloned the cDNA sequences of MMP-9gene on dairy goat to know its biological nature;analyzed the MMP-9gene expression in10tissuses on goat; analyzed the relationshipbetween the amount of goat milk somatic cell counts and the expression of MMP-9and theaffect of Staphylococcus aureus on the expression of MMP-9in goat mammary epithelialcells. These will provide a foundation for further research of the relationship between theexpression of MMP-9and mastitis.The results are as follows:(1) The MMP-9gene of dairy goat was cloned by application of RT-PCR and wasanalyzed by sequencing and bioinformatics.The results showed that the cloned MMP-9genefragment contained complete a2130bp CDs sequence (GenBank accession No.JQ670877),The gene encoded a protein containing709amino acids. The similarity of goat MMP-9cDNA sequence with that of human, mouse, and cattle were determined as84%,80%and96%, respectively, and the similarity of its amino acid sequence with that of human, mouseand cattle were determined as79%,73%and94%. The isoelectric point was5.23. It had asignal peptide sequence including19amino acid. The maximum hydrophilicity value is2.667and the maximum hydrophobicity value is3.444. It had35probably protein kinasephosphorylation sites. The probability of location in the extracellular is66.7%. The secondarystructure had random coil for58.25%, extension chain19.61%, α-helix14.67%, β-pleatedsheet7.48%. It also had a ZnMc domain and three FN2domains. (2) Using real-time quantitative PCR to test10tissuses about the MMP-9geneexpression on dairy goat. The results show that MMP-9gene is expressed in the fat, intestine,spleen, lung, breast, rumen, kidney, muscle, liver and heart tissuses. The expression level inthe spleen is the highest, then in the intestine and ammary gland, in the heart is the lowest.Analysis of variance showed a significant difference in the the spleen, intestine and ammarygland compared to other tissues(P＜0.05).(3) Study of the relationship between the expression level of MMP-9gene and somaticcell count. The results show that MMP-9gene expression increased with higher somatic cellcount, on the dairy goats in the same parity, same age and the same lactation. Taking MMP-9expression level as independent variables and the number of somatic cells as the dependentvariable, for group1and group2, the regression equation are as follows: y=0.6185Ln(x)+4.9009and y=0.5943Ln(x)+4.9095, R are0.9949(P＜0.05) and0.9953(P＜0.05).(4) Using staphylococcus aureus process dairy goat mammary epithelial cells to test theaffect on the expression of MMP-9gene in mammary epithelial cell in the consistency of103,105and107个/mL for2h,4h and6h. The results show that staphylococcus aureus can upregulate the expression of MMP-9gene. There is no significant difference between groupusing个to103processing2h and the control(P＞0.05), other groups also have significantdifference to the control (P＜0.05). |
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