fth1在b.ovis019株侵染小鼠巨噬細胞中的功能研究

1、石河子大學(xué)碩士學(xué)位論文FTH1在B.ovis 019株侵染小鼠巨噬細胞中的功能研究姓名：杜軍偉申請學(xué)位級別：碩士專業(yè)：預(yù)防獸醫(yī)學(xué)指導(dǎo)教師：王遠志20100601IIAbstract Object：Brucellosis is a zoontic disease caused by members of the genus Brucella. Brucella may parasite in macrophage and inhibit

2、cell apoptosis at long-term, which causes many organs damage and chronic infection for human and livestock. It is very important to explore the relation between brucella’s intracellullar mechanism and chronic infection.

3、The results of current etiologic study show that outer membrane proteins (OMPs) and type secretion Ⅳsystem (TFSS) proteins are putative virulent factors because these elements play an important role in brucella’s invasi

4、on, survival and multiplication. The function study of macrophage target proteins interacting with brucella virulent factors becomes molecular basis of brucella intracellullar parasite mechanism. This study is useful to

5、screen specific candidate drugs to brucellosis. Methods: (1)To verificate ferritin heavy chain 1(FTH1) interacted with brucella TFSS VirB5 protein by co-immunoprecipitation (Co-IP). (2)The target genes including FTH1,16S

6、 rRNA , GAPDH and apoptosis-related genes were cloned, whose Std and melt curve were made. (3)Three positive and one negative pSIREN-siRNAs targeted to FTH1 were transfected into mouse RAW264.7 macrophages with TurboFect

7、TM in vitro Transfection Reagent. The optimal pSIREN-siRNA were screened by detecting the expression of FTH1 by Real-time PCR. (4)The optimal pSIREN-siRNA was transfected into mouse RAW264.7 macrophages. After 48h the tr

8、ansfected macrophages were infected with B.ovis 019 strain[0] for 4h and the expressions of 16S rRNA and apoptosis-related genes were quantitated and compared by Real-time PCR. (5) The morphologic change of RAW264.7 macr

9、ophages infected with B.ovis 019 strain before RNAi and after RNAi were observed by electron microscope. (6) The cell culture fluid supernatant of Mouse RAW264.7 macrophages transfected with optimal pSIREN-siRNA and infe

10、cted with B.ovis 019 strain for 15min, 30min, 1h and 4h was detected by ELISA. Results: (1) The results of Co-IP showed that VirB5 interacted with FTH1. (2) The eleven target genes including FTH1,16S rRNA ,GAPDH and apop

11、tosis-related genes were cloned and their Std value were made. (3)Three positive siRNA plasmids were successfully transfected into mouse RAW264.7 macrophages. The optimal pSIREN-C was screened and its inhibition rate rea

12、ched to 98%. (4) The results of Real-time PCR showed that the capability of Brucella’s invasion was inhibited by 84% after RNAi while two apoptosis suppressor gene (Mkl1 and Birclb) expression were inhibited by 76.3% and