| Sumoylation is mainly involved in many kinds of cell function, such as cell cycle regulation, signal transduction, transcription regulation, apoptosis, cell proliferation and differentiation, transport of protein, DNA repair and regulation of stress reaction and so on. Brucella is one typical facultative intracellular bacterium that live and breed in the macrophages. Currently, the study about ubiquitin in bacteria-mediated cell biological processes is more less. The research on Brucella-mediated has important significance in the changes of SUMO and the fluence of apoptosis.This study mainly includes the following aspects:1. The SUMO1/2/3proteins were expressed in colibacillus, purificated and preparated of antibodyAccording to mice SUMO1/2/3gene published in GenBank, the sequence specific primers were designed. Prokaryotic expression vectors pET32a-SUMO1/2/3were successfully constructed and expressed in BL21(DE3) cells by SDS-PAGE. The result showed that A protein band about30KD was demonstrated, and the highest peak emerged at6h. the polyclonal antibodies by injection the purified SUMO1/2/3fusion protein to rabbits were acquired. By analysis of Western blot, it showed that the expressed protein has good immunogenicity.2. Effection on expression of SUMO1in cells infected by BrucellaWestern blotting and qRT-PCR was used to examined SUMO1protein expression level and SUMO1/2/3mRNA transcription levels of macrophage infected with Brucella M5-90and16M strains. In the study, the levels of SUMO1protein expression and mRNA transcription emerged that both originally ascended and then reduced during12h after macrophage infected with M5-90and16M strains, and SUMO1expression levels infected with M5-90andl6M strains peaked at4h (p<0.01) and8h (p<0.01), respectively. The levels of SUMO2mRNA transcription emerged that both originally ascended and then reduced during12h after macrophage infecting Brucella. The level of SUMO2mRNA transcription of macrophage infected with M5-90was overtop that with16M strains peaked at4h (p<0.01) and8h (p<0.01). The levels of SUMO3mRNA transcription of macrophage infected with M5-90is overtop the levels with16M strains and Control group.3. Construction of eukaryotic expression vector containing SUMO1and how it may affect the survive and cell apoptosis by BrucellaAccording to SUMO1and pcDNA3.1(-) gene sequences, the sequence specific primers were designed. prokaryotic expression vector pcDNA-SUMO1was successfully constructed by Identified and sequenced. Transfection conditions of pcDNA-SUMO1was confirmd plasmid By Real-time fluorescent quantitative method; Through the transient transfection of recombinant plasmid pcDNA-SUMO1enables SUMO1excessive expression in the cell, then detected the condition of Brucella intracellular survival in cells with Brucella M5-90and16M strains infecting by LgCFU method, the M5-90bacteria number in cells of transfecting pcDNA-SUMO1plasmid presented downward trend within12h, and was lower than Control group behind4h. the M5-90bacteria number in cells of transfecting pcDNA-SUMO1plasmid presented increased trend behind4h and was lower than Control group; Through the transient transfection of recombinant plasmid pcDNA-SUMO1enables SUMO1excessive expression in the cell, then detected the condition of cells apoptosis. The cells apoptosis level is all increased. The apoptosis cells of transfecting pcDNA-SUMO1plasmid is higher than other groups.The results show that:the vectors pET32a-SUMO1/2/3and pcDNA-SUMO1were successfully constructed, and prepared polyclonal antibody (rabbit-anti SUMO1/2/3). Brucella infected cells can cause the changes of SUMO1expression and transcription level of SUMO1/2/3, because SUMO1/2/3played a certain role in host cell survival of Brucella; SUMO1excessive expressed in macrophage what can inhibit Brucella intracellular survival ability and promote cells apoptosis level caused by Brucella. The study effect SUMO1act on such cell factor, and thus inhibit Brucella survival in cell and cells apoptosis with Brucella infecting. |
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