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嗜热毛壳菌端粒酶的表达纯化及酶学特性分析
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引用本文:尹 虎,宋泽玉,奚绪光.嗜热毛壳菌端粒酶的表达纯化及酶学特性分析[J].西北农业学报,2024,(3):532~541
DOI:10.7606/j.issn.1004-1389.2024.03.015
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作者单位
尹 虎,宋泽玉,奚绪光 (西北农林科技大学 生命科学学院陕西杨凌 712100) 
基金项目:国家自然科学基金(31870788)。
中文摘要:为进一步探究端粒酶的酶学特性,以嗜热毛壳菌(Chaetomium thermophilum, Thermo)端粒酶为研究对象,通过生物化学、生物信息学等方法得到4种不同长度的Thermo端粒酶蛋白,分别与体外转录得到的Thermo端粒酶RNA进行组装。通过端粒酶体外延伸试验检测组装体的反转录活性,进一步分析影响其反转录活性的各种因素。结果表明,4种不同长度的Thermo端粒酶蛋白重组质粒经大肠杆菌表达系统诱导表达后均可得到相应的Thermo端粒酶蛋白,且纯度均在90%以上;体外孵育试验表明,在10 mmol/L HEPES-KOH(pH 8.0)、1 mmol/L MgCl2、100 mmol/L NaCl、3 mmol/L KCl、体积分数25% Glycerol、10 units/μL Rnasin的条件下,端粒酶蛋白与RNA的摩尔浓度比达到1∶1及以上时,两者能有效复合;端粒酶体外延伸试验发现,结构完整的Thermo端粒酶组装体具有很强的反转录活性,可使18nt-DNA引物反复延伸。TEN结构域不完整并未减弱Thermo端粒酶组装体的反转录活性,说明其在反转录过程中未发挥显著作用。 T-PK区或TWJ区缺失的组装体均不能使18nt-DNA引物发生延伸,表明RNA结构的完整性是端粒酶发挥反转录功能的必要条件。
中文关键词:Thermo端粒酶  蛋白表达纯化  RNA体外转录  反转录活性
 
Expression and Purification of Telomerase from Chaetomium thermophilus and Analysis of Its Enzymatic Properties
Abstract:To further investigate the enzymatic properties of telomerase,using Chaetomium thermophilum (Thermo) telomerase as the object,four different lengths of Thermo telomerase proteins were obtained by biochemistry and bioinformatics,and assembled with Thermo telomerase RNA obtained by in vitro transcription.The reverse transcription activity of the assemblies was detected by telomerase in vitro extension assay,and various factors affecting their reverse transcription activity were further analyzed.The results showed that the recombinant plasmids of four different lengths of Thermo telomerase proteins could obtain the corresponding Thermo telomerase proteins after induction and expression by the E.coli expression system,and the purity of all of them was above 90%.The in vitro incubation assay showed that the molar concentration ratio of telomerase protein to RNA reached 1∶1 and under the conditions of 10 mmol/L HEPES-KOH (pH 8.0),1 mmol/L MgCl2,100 mmol/L NaCl,3 mmol/L KCl,25% Glycerol by volume,10 units/μL Rnasin above,the two can complex effectively;in vitro telomerase extension assay revealed that the structurally intact Thermo telomerase assemblies had strong reverse transcription activity and could make the 18nt-DNA primers extend repeatedly.The incomplete TEN domain did not diminish the reverse transcription activity of the Thermo telomerase assemblies,indicating that they did not play a significant role in the reverse transcription process.
keywords:Thermo telomerase  Protein expression and purification  RNA transcription in vitro  Reverse transcriptional activity
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