| 王瑞睿 1,2，马克异 3.罗氏沼虾 Doublesex 基因的原核表达与蛋白纯化[J].广东农业科学,2023,50(7):156-163 |
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罗氏沼虾 Doublesex 基因的原核表达与蛋白纯化 |
| Prokaryotic Expression of Doublesex Gene From Macrobrachium rosenbergii and Protein Purification |
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| DOI：10.16768/j.issn.1004-874X.2023.07.016 |
| 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基金项目（31902348） |
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| 中文摘要: |
| 【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，可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，将其转化大肠杆菌 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 检测发现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初步依据。 |
| 英文摘要: |
| 【Objective】Doublesex, a member of the DMRT gene family, plays a key role in controlling sex-specific differentiation. To clone the Open Reading Frame (ORF) of Macrobrachium rosenbergii Doublesex (MrDsx) gene, construct the recombinant plasmid and induce its expression, purify and obtain the recombinant protein MrDsx, will provides basic information for further study of MrDsx function. 【Method】Based on the ORF sequence of MrDsx, 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 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 analysis.【Result】Using the gonad cDNA as template, a single DNA band with the size of about 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 analysis 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 by prokaryotic expression of MrDsx. High purity active MrDsx protein can be obtained by the dissolution, purification and renaturation. This study provides a preliminary basis 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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