| 原核生物核糖核酸酶HII基因的克隆及在大肠杆菌中的高效表达 |
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| 引用本文： | 席慧,刘凤华,周霞,徐燕,李永海,高音.原核生物核糖核酸酶HII基因的克隆及在大肠杆菌中的高效表达[J].生物技术通报,2004(3):40-44. |
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| 作者姓名： | 席慧 刘凤华 周霞 徐燕 李永海 高音 |
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| 作者单位： | 首都师范大学生物系,北京,100037 |
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| 摘 要： | 核糖核酸酶HII (RNaseHII)能有效降解RNA和DNA杂交链中的RNA链。为进一步研究其功能 ,利用大肠杆菌XL1blue为模板 ,相应的寡聚脱氧核苷酸为引物 ,PCR扩增大肠杆菌RNaseHII(rnh 2 )基因 ,并将目的基因连接到克隆载体 pUC18上 ,经测序确认无误 ,分别亚克隆到能够进行IPTG诱导的表达载体pTrcHisC和进行温度诱导的表达载体pBV2 2 0上。重组质粒转化到大肠杆菌DH5α细胞中获得高效表达。在载体pTrcHisC和 pBV2 2 0中目的蛋白RNaseHII的表达量均超过菌体总蛋白的 2 0 % ,且表达产物以稳定的包涵体形式存在。此项工作为以后目的蛋白的纯化提供了有利条件 ,并为研究其结构和功能奠定了基础。
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| 关 键 词： | 核糖核酸酶H 核糖核酸酶HII 诱导表达 包涵体 |
Cloning and Overexpression of Prokaryotic Ribonucleic acid enzyme HII in Escherichia coli |
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| Xi Hui,Liu Fenghua,Zhou Xia,Xu Yan,Li Yonghai,Gao Yin.Cloning and Overexpression of Prokaryotic Ribonucleic acid enzyme HII in Escherichia coli[J].Biotechnology Bulletin,2004(3):40-44. |
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| Authors: | Xi Hui Liu Fenghua Zhou Xia Xu Yan Li Yonghai Gao Yin |
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| Abstract: | By using the Polymerase Chain Reaction (PCR) technique, the 594 bp DNA fragment of ribonucleic acid enzyme HII gene (rnh 2) was amplified from the Escherichia coli XL1blue cell. The gene was inserted into expression vector pTrcHisC and pBV220 after it was cloned into plasmid pUC18 for sequencing. Protein expression was induced by IPTG or temperature. SDS-PAGE confirmed that the molecular weight of the recombinant protein was 28 or23 kDa in the form of insoluble inclusion body and the recombinant protein accounted for above 20% of the total cell protein. |
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| Keywords: | Ribonucleic acid enzyme H (RNase H) Ribonucleic acid enzyme HII(RNase HII) Induced expression Inclusion body |
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