The Effectiveness of GSL in Brucella Ovis 019 Strain Infecting Mouse Macrophage
|Course||Basic Veterinary Science|
|Keywords||Lectin gene RAW264.7 macrophage Brucella ovis 019 strain RNAi|
Object:Brucellosis is a zoonotic disease caused by members of the genus Brucella. It is widely popular in the world, especially in developing countries. Brucella is a facultative intracellular parasitic bacterium. Bacteria into macrophages not only kill but will not be protected, as this is the main reason patients can not be cured. It is useful to describe brucella and prey proteins of macrophages. This article chosen and sheep Brucella outer membrane protein interacting macrophages GSL gene, Fragments through the design of shRNA interference filter GSL normal gene expression, through the use of sheep Brucella 019 strain infection, and further explore the GSL gene function, to explore the intracellular Brucella parasitism basis.Methods:(1)Published by the macrophage GSL gene nucleic acid sequences of primers designed according to the principle of RNAi, siRNA designed three specific positive and a negative siRNA molecules of molecular construct shRNA expression vector, named pSI-L0, pSI-L17, pSI-L29, pSI-L34; (2)The siRNAs were transfected into RAW264.7 cells and vena caudalis of BABL/C mouse. The inhibition effect was evaluated both in RAW264.7 cells andexperimental mouse by using Real-time PCR and immunohistochemistry. (3)Brucella ovis 019 strain infection vector transfected interference before and after the RAW264.7 macrophages, with real-time quantitative PCR, ELISA, electron microscopy methods, respectively, both from genetic level, cell factor, morphology the detection of Brucella The relative internal standard gene 16SrRNA expression and morphological changes, by comparing the interference vector before transfection Brucella ovis 019 strain to determine the relative number of genes in the RAW264.7 cells infected with the process of relevance, and analysis of GSL gene in Brucella ovis 019 strain infection in RAW264.7 macrophage function.Results:The restriction map and sequencing confirmed, The constructed GSL shRNA expression vector containing the gene fragment of sequence upstream and downstream regulatory elements express complete; by the cell level in transfected 500ng pSI-L17, pSI-L29, pSI-L34 plasmid could effectively inhibit the target gene in RAW264.7 cells, inhibition rate of up to 83.02%. The intravenous injection of 5μg the siRNA molecules of different gene expression in mice GSL has some inhibitory effect, the inhibition rate of between 84-96%, so the most efficient filter out interference vector pSI-L34; immunohistochemistry monitoring, that fragments can interfere with a marked decrease in GSL spleen expression. With 019 at different time periods of the transfected plasmid pSI-L34 macrophages infected with the infection time longer, the number of bacteria into the cells significantly increased, indicating GSL genes and Brucella infection RAW264.7 cells related; use of cytokines kit in cell supernatant cytokine levels can be seen, inflammatory cytokines IL-6, TNF-αand has its inflammatory symptoms; nucleus can be seen with electron microscopy significant of engorgement, that when GSL gene silencing were infected with 019, macrophages appeared necrosis.