凝集素在綿羊種布魯氏菌O19株侵染小鼠巨噬細(xì)胞中的作用.pdf

1、石河子大學(xué)碩士學(xué)位論文凝集素在綿羊種布魯氏菌O19株侵染小鼠巨噬細(xì)胞中的作用姓名：葛陽春申請學(xué)位級別：碩士專業(yè)：基礎(chǔ)獸醫(yī)學(xué)指導(dǎo)教師：陳創(chuàng)夫;王遠(yuǎn)志20100601IIAbstract Object：Brucellosis is a zoonotic disease caused by members of the genus Brucella.It is widely popular in the world, especially i

2、n 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 t

3、o 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

4、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：⑴Published by the macrophage GSL gene nu

5、cleic 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, p

6、SI-L29, pSI-L34; ⑵The 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 immunohisto

7、chemistry. ⑶Brucella 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 genet

8、ic 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 str

9、ain 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 res

10、triction 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 5

11、00ng 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

12、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 dec

13、rease 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, in

14、dicating 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-α and has its inflammatory symptoms; nucleus can be