Studies On Construction Of Transgenic Cell Line With The Resistance To Brucella Melitensis And Its Relative Issues

Brucellosis is a kind of zoonosis disease that affects a broad range of mammals, including livestock and humans, Brucella melitensis is the causative agent of Brucellosis, which belongs to the Gram-negative, facultative intracellular parasites that survives and multiplies inside host macrophages. Recent studies have showed that during the infection process, the cellular prion protein (PrPc) and the class A scavenger (SR-A) receptor have been proposed as receptors for binding surface-exposed Hsp60 and Brucella LPS respectively, lipid-raft mediated entry is required for bacterial initial survival. Some kinds of downstream inflammatory / effector cytokine expression such asβ-defensin can be suppressed by inhibiting the immune response-related signaling pathways which at last result in the Brucella-related disease. According to the mechanisms of the Brucella infection, we had cloned SR-A receptor, selected appropriate antibacterial peptide, constructed the eukaryotic expression vector which has the ability to express chimeric peptide and established the transgenic cell lines. And meanwhile, related detects using molecular methods such as RT-PCR, Western Blot were performed in this study.1. RNA was isolated from the spleen of the sheep, and turned into the form of cDNA though the reverse reaction. Primers were designed by using the sequence of the cDNA of cattle, which can be found in GeneBank. Then the cDNA of sheep SR-A was obtained, and at last the cDNA of its ectodomain; The artificial sequence of a kind of antibacterial peptides called V13KL was got though the method of chemical synthesis, and 60bp signal peptide sequence was added in front of the cDNA of V13KL; The cDNA of full-length sheepβ-defensin was obtained though the method of gene splicing by overlap extension PCR(SOE PCR) by using three pairs of long primers; Then a new kind of chimeric gene was spliced though SOE PCR by using three parts: the cDNA ofβ-defensin, SR-A ectodomain and linker; The cDNA ofβ-defensin and V13KL were cloned into the pIRES2-EGFP vector, forming the recombinant plasmid , and then the cDNA of defensin-linker-SR-A ectodomain was cloned into the two vectors pcDNA3.1 and pEF1α-GFP, forming the recombinant plasmid. 2. For transient transfection, the FuGene HD transfection Reagent:DNA ratio(μl FuGENE HD:μg DNA)was 6:2, and the complex incubation time was 6-8h, which can meet the optimal result; By transfecting the two expressing vectors into COS-7 cells for transient transfection, the peptides of interest can be expressed in COS-7 cells, and related influence to the physiological functions such as the ratio of cell necrosis and apoptosis can be detected by using flow cytometer(FCM)after PI/Hochest33342 staining. The results showed that the ratios of apoptosis of COS-7 cells which can express V13KL andβ-defensin were lower than those of the controls, the difference was significant; Meanwhile, ratios of apoptosis of COS-7 cells which can expressβ-defensin was lower than that of the COS-7 cells which can express V13KL but there is no significant difference; The vectors were then injected into the pronucleus of the mouse embryos, and the results showed that there was no significant difference in the developmental rate of blastula and GFP positive rate when compared with the control group (empty vector). This result indicated that the antibacterial peptides can be harmless to the early embryos.3. COS-7 cells were transfected with the plasmid of pEF1α-defensin-SR-A ectodomain -GFP, and the total proteins in COS-7 cells were collected for the tests of Western Blot. The results showed that the designed chimeric peptide can be efficiently expressed in this COS-7 cell system; And 650ug/ml is the optimal concentration of G418 for selecting the transgenic cell line which can express this kind of chimeric peptide; After the beginning of the transfection with FuGENE HD, selection using G418, after 14 days, apparente cell clones have been detected, and then they were picked, passaged and frozen. Genome and RNA from these cell clones were extracted and detected, the results showed that the chimeric gene has integrated into the genome of transgenic cell lines and the RNA of which have been transcripted.