Tat-PTD介导的金属硫蛋白高产量表达及其高效表达重组菌株重金属抗逆性研究

中国生物工程杂志

2013, Vol. 33

Issue (9): 17-23

研究报告

Tat-PTD介导的金属硫蛋白高产量表达及其高效表达重组菌株重金属抗逆性研究

李华玲, 王凯, 秦艳, 刘丹丹, 陈文飞

扬州大学医学院扬州 225001

Tat-PTDs Mediated Metallothionein High-yield Expression in Escherichia coli and Its Heavy Metal Resistance Studying

LI Hua-ling, WANG Kai, QING Yan, LIU Dan-dan, CHEN Wen-fei

Medical College of Yangzhou University, Yangzhou 225001, China

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摘要： 目的:探讨Tat-PTDs对金属硫蛋白(metallothionein,MT)表达的促进作用及其体外高效表达重组菌株的重金属抗逆性。方法:采用聚合酶链式反应从小鼠肝脏组织中克隆了金属硫蛋白 MT-1和MT-2 基因的编码序列,进而分别亚克隆入原核表达质粒pET28b-Tat和pET28b,测序验证后转化入大肠杆菌BL21(DE3)细胞,采用1mmol/L诱导剂IPTG诱导表达,并于诱导表达0h,2h,4h,6h时收集菌体,超声破碎后制备总蛋白、上清蛋白和沉淀蛋白并进行SDS-PAGE和Western blot鉴定。针对上述诱导表达4hrs的菌液适量稀释后转接入新的LB培养基,采用分光光度仪测定高效表达金属硫蛋白菌株对5mmol/L铜的抗逆性。结果:Tat-PTDs可明显增加金属硫蛋白的表达产量,且Tat融合蛋白主要为可溶性表达;Western blot及其柱状图分析结果显示,Tat融合蛋白的表达水平约为非Tat融合蛋白的5~8倍;高效表达Tat-MT-1和Tat-MT-2蛋白的菌株具有明显的抗重金属铜的活性,与非Tat标签融合蛋白和空载对照组相比存在显著性差异(P<0.01)。结论:实验数据表明,Tat-PTDs蛋白转导技术可以促进小鼠金属硫蛋白基因在原核生物中的可溶性高表达,Tat融合蛋白的表达水平比非Tat融合蛋白的表达有显著提高,其克隆菌株对重金属铜活性的抗性也有明显提高。

关键词： Tat-PTDs; 可溶性; 重金属抗逆性

Abstract: Objective:To explore the feature of Tat-PTDs for metallothionein (MT) high-yield expression in E. coli and its heavy metal resistance in vitro. Methods:the MT-1, 2 encoding genes in Balb/c mice liver tissue was amplified using polymerase chain reaction (PCR) initially, and subcloned into the prokaryotic expression plasmid pET28b-Tat and pET28b followed direct sequencing separately. The validated plasmids were transformed into E. coli BL21 (DE3) cells and induced using 1 mmol/L IPTG, and the protein samples at 0 h, 2 h, 4 h and 6 h were prepared and identified by SDS-PAGE and Western blot. Subsequently, the bacterial cells at 4 h induced were diluted using LB medium and study its resistance of 5 mm copper. Results:This study demonstrated that Tat-PTDs could obviously increase the expression of MT-1, 2 in E. coli and Tat fusion protein mainly expressed in the bacterial supernatant. The Tat fusion protein expression level was 5~8 folds compared to its control according to the results of Western blot and histogram analysis. Furthermore, the over-expressed Tat-MT-1, 2 bacterial cells had an obvious resistance to 5mm copper compared with its control (P<0.01). Conclusion:the data showed that protein transduction based on Tat-PTDs can promote genes of mouse metallothionein high-yield and soluble expression in prokaryotic system. The Tat fusion protein expression level was significantly improved compared to non-Tat fusion protein and its resistance to copper was also increased.

Key words: Tat-PTDs Solubility Heavy metal resistance

收稿日期: 2013-06-20 出版日期: 2013-09-25

ZTFLH:

Q815

基金资助: 国家自然科学基金资助项目(81100862)

通讯作者: 李华玲hlli@yzu.edu.cn E-mail: hlli@yzu.edu.cn

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引用本文:

李华玲, 王凯, 秦艳, 刘丹丹, 陈文飞. Tat-PTD介导的金属硫蛋白高产量表达及其高效表达重组菌株重金属抗逆性研究[J]. 中国生物工程杂志, 2013, 33(9): 17-23.

LI Hua-ling, WANG Kai, QING Yan, LIU Dan-dan, CHEN Wen-fei. Tat-PTDs Mediated Metallothionein High-yield Expression in Escherichia coli and Its Heavy Metal Resistance Studying. China Biotechnology, 2013, 33(9): 17-23.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I9/17

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