| 摘要: |
| [目的]证实蓝细菌Anabaena sp.PCC 7120复制酶中分离的DnaE内含肽的自我剪接活性.[方法]分别将Anabaena PCC 7120中分离的2个dnaE基因的部分序列克隆到表达载体上,获得重组表达质粒,经超量表达和纯化后,免疫家兔获得相应的抗体;同时,两段分离的dnaE序列被克隆到同一表达载体,构建双重组表达质粒,两段序列具有各自的ORF,但由同一启动子调控.双重组表达质粒经诱导之后,利用以上获得的抗体对含有内含肽的DnaE蛋白在大肠杆菌表达体系中的自我剪接反应进行检测.[结果]在双重组表达质粒的表达产物中,抗体不仅可以分别与对应的蛋白质反应,而且还可以同时识别1条新条带,该条带的表观分子量与成熟DnaE的理论值大致相符.[结论]蓝细菌分离的DnaE内含肽在大肠杆菌表达体系中可以进行自我剪接;本研究中获得的抗体具有较好的特异性,无明显背景干扰,而且效价高,抗体可以稀释1000倍以上使用,也可以用于相关方面的研究. |
| 关键词: DnaE内含肽 重组表达质粒 自我剪接 蓝细菌 |
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| 基金项目:西北农林科技大学人才基金? |
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| Study on self-splicing of split intein of the DNA polymerase DnaE in cyanobacteria |
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| Abstract: |
| 【Objective】 The double recombinant expression plasmid was cloned to determine the self-splicing activity of split inteins located in split DnaE of cyanobacteria Anabaena sp.PCC 7120.【Method】 The corresponding antiserum against the two split DnaE precursor proteins were collected by injecting rabbits with purified proteins after over-expression from reconstructing recombinant expressing plasmid in Escherichia coli.In heterogeous E.coli,the antibodies were available to test the self splicing of split DnaEs.【Result】 Results showed that both precursor proteins and mature proteins were detected in E.coli and antibodies could only react with their corresponding proteins.【Conclusion】 The results suggested that trans-splicing existed between the split DnaEs which contain the split inteins.The antibodies were specific without odvious background disturbance and had high effect with applied concentration low to 1/1 000,so it could be used in related research. |
| Key words: DnaE intein recombinant expressing plasmid self splicing cyanobacteriun |
