CDNA Library Construction For Brucella Infected Macrophage And Interacting Proteins Screening And Identification

Brucellosis, which is caused by Brucella, is one of the most important bacterialzoonosis endemic in many countries, especially in developing countries. In2000years,all data suggest that worldwide economic losses due to brucellosis are extensive notonly in animal production but also in public health.The most critical problem that controlling and eradicating brucellosis in animalsand human beings is that the investigation in its pathogenic mechanism is not fullyclear. Brucella spp. are not found as a free living or commensal organisms. Thepreferred ecological niche for the brucellae is within the phagosomal compartment ofhost macrophages, and the capacity of the pathogen to establish and maintain chronicinfections is dependent upon its ability to survive and replicate within thesephagocytic cells. Experimental evidence indicates that the production of reactiveoxygen intermediates (ROIs) represent one of the primary mechanisms utilized byhost macrophages for limiting the intracellular replication of the brucellae.Superoxide dismutase (SOD) could detoxify these ROIs and help the brucellaeavoid oxidative killing, which would allow them to establish and maintain residencein their intracellular niche. In the course of brucella Infection， the superoxidedismutase secreted by B．melitensis16M how to intact within the macrophage protein,inhibit the oxidation mechanism of the cell, ease to the ingress of virulence factorsinto the host and Play effect is not clear. So in the research superoxide dismutase wasused as a bait Protein to search its interacted Protein from mouse macrophage cDNAlibrary using the yeast two hybrid system to explore its function in brucella infection.The titer of the primary cDNA library was1.8×10~8cfu/ml with a recombination rateof99%. PCR amplification of randomly picked clones revealed that the insertedcDNA fragments ranged from500to2000bp which reflected that the size distribution of the cDNA was convergence, which meets the wants of built library.The whole ORF length objective gene including SOD was cloned, sequenced andinserted into pSos vector to become bait plasmids. The bait plasmid was transformedinto cdc25Hα and further examined wether all bait plasmids can activate transcriptionof down-stream report gene in singles. The library was screened by co-transformationof the pSos-SOD bait constructed inio a temperature-sensitive cdc25Hα yeaststrain.The positive target plasmid was sequenced.The result was sent to NCBI forBLAST searches online.In the study, one positive target plasmid received from the mouse macrophagecDNA library. The gene is identical to the Mus musculus ribosomal protein S5(Rps5).SOD may be attracted to the mouse ribosomal S5(rpS5) which may provide energyand places. superoxide dismutase(SOD) detoxify these ROIs and help the brucellaeavoid oxidative killing,which would allow them to establish and maintain residencein their intracellular niche.