【作者】 潘滨；

【导师】 周雪平；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 双生病毒是一类世界范围内广泛发生的单链环状DNA病毒,主要以滚环复制的方式进行病毒基因组的复制。与此类病毒致病性紧密相关的新型卫星分子DNAβ依赖病毒DNA-A进行复制。为了进一步了解DNAβ复制的机制,本论文对烟草曲茎病毒(Tobacoo curly shoot virus, TbCSV)的复制相关蛋白基因(replication-associated protein,Rep)进行了原核表达,并对Rep的核酸结合特性进行了初步研究。 为了提高TbCSV Rep在大肠杆菌(Escherichia coli)中表达量,我们研究了大肠杆菌菌株、温度、诱导时间以及诱导剂IPTG终浓度等不同条件对GST-Rep融合蛋白表达量的影响。结果表明,大肠杆菌菌株Rosetta在37℃培养3hr后,使用终浓度为0.5mmol/L的IPTG在28°C诱导培养4hr时,GST-Rep融合蛋白表达量最高,表达的GST-Rep融合蛋白可占细菌总蛋白的42%。用GST-Sepharose 4B亲和层析柱对GST-Rep融合蛋白进行纯化,SDS-PAGE表明GST-Rep融合蛋白大小约为66 kDa,与预计的分子量大小一致,Western blot分析表明其能与GST多克隆抗体起特异性反应。Rep基因的优化表达为研究该基因的作用机理打下基础。 双生病毒编码的Rep具有结合核酸的能力。为了验证Rep能否结合DNAβ,我们利用TbCSV-Y35 Rep蛋白进行了核酸结合试验。以全长TbCSV-Y35 DNAβ分子作为探针,结果表明TbCSV-Y35 Rep蛋白能结合单、双链DNAβ。更多还原

【Abstract】 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.更多还原