| Geminiviruses are a group of plant viruses characterized by their unique twinned particle, and they use a rolling circle replication system to amplify their genomes. Novel satellite molecule named DNAβ was shown to be associated with geminiviruses and to be essential for induction of characteristic symptoms, and DNAp relys on help virus for replication. To further know the mechanism of DNAp replication, the replication-associated protein gene (Rep) of Tobacco curly shoot virus (TbCSV) was expressed in Escherichia coli and its nucleic acid binding ability was studied in this thesis.In order to improve the expression level of TbCSV Rep in Escherichia coli, the effects of various factors on the protein expression were studied, these factors included bacterial strain, culture temperature, inducing time, and the final concentration of inductor IPTG etc. The results indicated that the expression level of GST-Rep fusion protein reached highest using E. coli strain Rosetta and induced with 0.5 mmol/L IPTG as final concentration for 4 hours at 28℃ after 3 hours' culture at 37℃, and the expressed GST-Rep fusion protein accounted for 42% of the total cell lysates with the above culture conditions. The GST-Rep fusion protein was purified with GST-Sepharose 4B affinity chromatography, SDS-PAGE analysis showed that the molecular mass of GST-Rep fusion protein was about 66 kDa, and Western blot analysis indicated the GST polyclonal antibody could specifically bound to purified GST-Rep fusion protein. The optimized expression conditions of Rep will be useful for future research on function of Rep.Rep encoded by geminiviruses has nucleic acid binding ability. In order to test whether Rep can bind to DNAp, electrophoresis mobility shift assay (EMSA) was employed. The results showed that TbCSV-Y35 Rep fusion protein could bind double- and single-stranded DNA℃ when use whole TbCSV-Y35 DNAβ as probe.
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