| 王瑞睿,马克异.罗氏沼虾(Macrobrachium rosenbergii)Doublesex基因的原核表达与蛋白纯化[J].广东农业科学,2023,(7):- |
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罗氏沼虾(Macrobrachium rosenbergii)Doublesex基因的原核表达与蛋白纯化 |
| Prokaryotic Expression and Purification of Macrobrachium rosenbergii Doublesex Gene |
| 投稿时间：2023-04-28 修订日期：2023-08-24 |
| DOI： |
| 中文关键词: 罗氏沼虾 Doublesex 原核表达 蛋白免疫印迹 重组蛋白 透析复性 质谱分析 |
| 英文关键词: Macrobrachium rosenbergii Doublesex prokaryotic expression Western blot recombinant protein dialysis renaturation mass spectrometry |
| 基金项目:国家自然科学基金青年(31902348) |
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| 中文摘要: |
| 摘 要：【目的】Doublesex属于DMRT基因家族成员之一，在控制性别特异性分化中起关键作用。克隆罗氏沼虾（Macrobrachium rosenbergii）Doublesex基因（MrDsx）的开放阅读框序列，构建重组质粒并诱导其表达，纯化获取罗氏沼虾重组蛋白MrDsx，为后续开展罗氏沼虾MrDsx基因的功能研究提供基础数据。【方法】基于罗氏沼虾MrDsx的开放阅读框序列，通过特异PCR产物与表达载体pET-32a（+）连接，获得重组质粒pET-32a-MrDsx；将重组质粒pET-32a-MrDsx转化大肠杆菌 BL21（DE3）感受态细胞，构建罗氏沼虾MrDsx基因的原核表达细胞。利用异丙基-β-D-硫代半乳糖苷（IPTG）对阳性转化细胞进行诱导表达，并以SDS-PAGE电泳，Western blot和质谱分析检测诱导表达的重组蛋白。【结果】以罗氏沼虾性腺cDNA为模板，利用MrDsx基因的特异引物，PCR扩增获得了约660 bp大小的单一DNA条带。经测序确认为MrDsx基因的ORF序列。重组质粒pET-32a-MrDsx转化后的感受态细胞经IPTG在37 ℃条件下诱导4 h后可获得罗氏沼虾重组蛋白MrDsx。当IPTG终浓度为0.1~0.5 mmol/L时，重组蛋白MrDsx的表达量可达到峰值。可溶性分析显示，重组蛋白MrDsx主要以包涵体形式表达，经8 mol/L尿素溶解，Ni柱纯化及透析复性后，SDS-PAGE电泳和Western blot检测重组蛋白的相对分子量约55 kD，且条带单一，表明重组蛋白MrDsx能被鼠抗His-tag单克隆抗体特异性识别。质谱分析结果显示片段化肽段与理论的氨基酸序列相符，匹配度达到100%。【结论】成功获得了MrDsx基因原核表达的重组质粒pET-32a-MrDsx。诱导表达的重组蛋白经溶解、纯化及复性后，可形成高纯度的活性罗氏沼虾MrDsx重组蛋白，为后续开展罗氏沼虾MrDsx基因的功能研究、互作蛋白的筛选和性别调控机制的解析提供基础资料。 |
| 英文摘要: |
| Abstract: 【Objective】Doublesex, a member of the DMRT gene family, plays a key role in controlling sex-specific differentiation. This study, cloned the Open Reading Frame (ORF) of Macrobrachium rosenbergii Doublesex gene (MrDsx), constructed the recombinant plasmid and induced its expression, purified and obtained the recombinant protein MrDsx, will provides basic information for further study of MrDsx function. 【Method】Based on its ORF sequence of M. rosenbergii, the recombinant plasmid pET-32a-MrDsx was obtained by ligating the specific PCR product with the expression vector pET-32a (+). The recombinant plasmid pET-32a-MrDsx was transformed into the competent cells of Escherichia coli BL21 (DE3) to construct the prokaryotic expression cells of M. rosenbergii MrDsx. The isopropyl-beta-D-thiogalactoside (IPTG) was used to induce the expression of positive transformed cells, and the induced recombinant protein was detected by SDS-PAGE, Western blot and mass spectrometry. 【Result】Using the gonad cDNA as template, a single DNA band with the size of ~660 bp was obtained by the specific PCR primers of MrDsx. And the MrDsx ORF sequence was confirmed by sequencing. The recombinant protein MrDsx could be obtained from the transformed competent cells after induction by IPTG at 37℃ for 4 h. When the final concentration of IPTG was 0.1-0.5 mmol/L, the expression level of recombinant protein MrDsx reached its peak. Soluble analysis showed that the recombinant protein MrDsx was mainly expressed in the form of inclusion bodies. After solubilization with 8 mol/L urea, Ni purification, and dialysis renaturation, a single band was detected at the relative molecular weight of 55 kD by SDS-PAGE and Western blot, indicating that the recombinant protein MrDsx could be specifically recognized by His-tag mouse monoclonal antibody. The results of mass spectrometry showed that the fragmented peptide was consistent with the theoretical amino acid sequence, and the matching degree was 100%. 【Conclusion】The recombinant plasmid pET-32a-MrDsx was successfully obtained for prokaryotic expression of MrDsx. High purity active MrDsx protein can be obtained by the dissolution, purification and renaturation. This study provides basic information for the subsequent study of MrDsx gene function, screening of interacting proteins and elucidation of sex regulation mechanisms in M. rosenbergii. |
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