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| 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 |
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| Abstract: |
| 【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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