青海血蜱Hq012基因在大肠埃希菌中的表达与纯化

doi:10.11853/j.issn.1003.8280.2023.06.001

中国媒介生物学及控制杂志 ›› 2023, Vol. 34 ›› Issue (6): 713-718.DOI: 10.11853/j.issn.1003.8280.2023.06.001

• 实验研究 • 下一篇

青海血蜱Hq012基因在大肠埃希菌中的表达与纯化

马婧^1,2, 陈开廷², 广慧², 赵文斌², 杨寅冉², 高金亮^1,2, 曹美娜²

1. 内蒙古医科大学鄂尔多斯临床医学院, 内蒙古鄂尔多斯 017000;
2. 鄂尔多斯市中心医院分子医学实验室, 内蒙古鄂尔多斯 017000

收稿日期:2023-04-12 出版日期:2023-12-20 发布日期:2023-12-26
通讯作者: 高金亮,E-mail：lmm20150311@163.com;曹美娜,E-mail：caomeina2009@hotmail.com
作者简介:马婧，女，在读硕士，主要从事蜱源抗凝血物质的研究，E-mail：514093408@qq.com
基金资助:
国家自然科学基金（81760375）；内蒙古自治区自然科学基金（2021MS08063）；内蒙古医科大学“科技百万工程”联合研究项目[YKD2020KJBW（LH）047]

Expression and purification of Hq012 gene of Haemaphysalis qinghaiensis in Escherichia coli

MA Jing^1,2, CHEN Kai-ting², GUANG Hui², ZHAO Wen-bin², YANG Yin-ran², GAO Jin-liang^1,2, CAO Mei-na²

1. Ordos Clinical Medical College of Inner Mongolia Medical University, Ordos, Inner Mongolia 017000, China;
2. Laboratory of Molecular Medicine, Ordos Central Hospital, Ordos, Inner Mongolia 017000, China

Received:2023-04-12 Online:2023-12-20 Published:2023-12-26
Supported by:
National Natural Science Foundation of China (No. 81760375); Natural Science Foundation of Inner Mongolia Autonomous Region of China (No. 2021MS08063); Joint Research Project of "Science & Technology Million" Project of Inner Mongolia Medical University (No. YKD2020KJBW[LH]047)

摘要/Abstract

摘要： 目的优化青海血蜱重组Hq012蛋白（rHq012）包涵体的诱导表达和纯化条件。方法从青海血蜱cDNA表达文库中克隆到1个在GenBank中无同源序列的新基因Hq012,构建原核表达质粒pET-30a-Hq012并转化大肠埃希菌感受态细胞[Escherichia coli Rosetta（DE3）],用异丙基硫代-β-D-半乳糖苷（IPTG）诱导表达rHq012。高压均质机裂解细菌,用6 mol/L盐酸胍溶解包涵体。比较先复性后纯化和先纯化后复性2种方法所得目的蛋白的产量。先复性后纯化即用稀释法复性后再用镍离子亲和层析法纯化蛋白；先纯化后复性即用镍离子亲和层析法纯化后再进行透析法复性。用12%十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定。结果 Hq012基因的核苷酸序列长度为759 bp,包含1个486 bp的开放阅读框,编码一个相对分子质量为18 500的蛋白质。诱导表达以浓度为0.1 mmol/L的IPTG诱导8 h蛋白表达量最高,包涵体用6 mol/L盐酸胍溶解。与先纯化后复性相比,先复性后纯化收获的rHq012产量相对较高。镍离子亲和层析纯化后的蛋白质相对分子质量约为17 000,与预期的结果一致。结论 0.1 mmol/L IPTG诱导8 h是青海血蜱rHq012蛋白的最佳诱导条件,先复性后纯化的方法是蜱源重组蛋白rHq012更优的纯化方法,研究为包涵体蛋白的纯化提供了参考。

关键词: 蜱源蛋白质, 包涵体, 复性, 纯化

Abstract: Objective To optimize the conditions for the induction expression and purification of recombinant Hq012 (rHq012) inclusion bodies of Haemaphysalis qinghaiensis.Methods The novel gene Hq012 with no homogenous sequences in the GenBank database was cloned from the cDNA expression library of H. qinghaiensis to be transfected into Escherichia coli Rosetta (DE3) using the prokaryotic expression plasmid pET-30a-Hq012. Isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of rHq012 protein. After homogenizing the bacteria under high pressure, the inclusion bodies were dissolved in 6 mol/L guanidine hydrochloride solution. We compared the yield of the target protein obtained by first renaturation and then purification (refolding by dilution followed by purification with nickel ion affinity chromatography) and by first purification and then renaturation (purification with nickel ion affinity chromatography followed by refolding by dialysis). The obtained protein was identified by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis.Results The nucleotide sequence length of the Hq012 gene was 759 bp, containing an open reading frame of 486 bp, encoding a protein with a relative molecular mass of 18 500. The protein expression was the highest at 8-hour induction with IPTG at 0.1 mmol/L. The inclusion bodies were dissolved with 6 mol/L guanidine hydrochloride. The yield of rHq012 harvested by first renaturation and then purification was higher compared with that by first purification and then renaturation. The relative molecular mass of the protein purified by nickel ion affinity chromatography was about 17 000, which was consistent with expected results.Conclusions Induction at an concentration of 0.1 mol/L IPTG for 8 hours was the best induction condition, and the method of first refolding followed by purification was an optimal purification method for the tick-derived recombinant protein rHq012. The study provides a reference for the purification of inclusion body protein.

Key words: Tick-derived protein, Inclusion body, Renaturation, Purification

中图分类号:

马婧, 陈开廷, 广慧, 赵文斌, 杨寅冉, 高金亮, 曹美娜. 青海血蜱Hq012基因在大肠埃希菌中的表达与纯化[J]. 中国媒介生物学及控制杂志, 2023, 34(6): 713-718.

MA Jing, CHEN Kai-ting, GUANG Hui, ZHAO Wen-bin, YANG Yin-ran, GAO Jin-liang, CAO Mei-na. Expression and purification of Hq012 gene of Haemaphysalis qinghaiensis in Escherichia coli[J]. Chinese Journal of Vector Biology and Control, 2023, 34(6): 713-718.

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青海血蜱Hq012基因在大肠埃希菌中的表达与纯化

Expression and purification of Hq012 gene of Haemaphysalis qinghaiensis in Escherichia coli

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