Paralichthys olivaceus male related Dmrt1 recombinant protein and application thereof

A technology of recombinant protein and flounder, applied in the field of molecular biology, can solve the problems of not allowed to use, unsuitable for application, high cost of use, etc.

Pending Publication Date: 2022-07-15

INST OF OCEANOLOGY - CHINESE ACAD OF SCI

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AI-Extracted Technical Summary

Problems solved by technology

Sex hormones and their receptor inhibitors are not allowed to be used in aquaculture because they will pollute the water environment; aromatase inhibitors are not allowed due to...

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to paralichthys olivaceus Dmrt1 recombinant protein and application. The paralichthys olivaceus Dmrt1 recombinant protein is obtained for the first time by utilizing an in-vitro recombinant expression technology, a preparation method of the recombinant protein is provided, and the paralichthys olivaceus gonad tissue is incubated by utilizing the recombinant protein, so that the paralichthys olivaceus Dmrt1 recombinant protein has biological activity. The recombinant Dmrt1 protein is injected into an abdominal cavity of a female adult paralichthys olivaceus, so that cell nucleus chromatin of an oocyte in an ovarian tissue of the female adult paralichthys olivaceus is condensed. Further injecting the vaccine into the abdominal cavity of the fry with undifferentiated gonad to respectively cause the obvious decrease and increase (plt) of the estrogen and androgen levels in the juvenile fish body; 0.05), and the male rate is increased from 0% to 82%. The recombinant protein can be used for sex control of fishes such as paralichthys olivaceus, and a new method and way can be provided for sex control and unisexual production in fish culture.

Application Domain

Microorganism based processesPisciculture and aquaria +2

Technology Topic

OocyteGonad tissue +14

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Examples

Experimental program(5)

Example Embodiment

[0030] Example 1 Prokaryotic recombinant expression of flounder Dmrt1 protein

[0031] 1. Construction of recombinant plasmid Blunt E2-Dmrt1 and recombinant strain

[0032] Extracting the RNA of the flounder testis tissue, and reverse transcribing it into cDNA, using PCR to amplify the entire coding region of the flounder Dmrt1, the encoded amino acid sequence is shown in SEQ ID NO: 1, and then using the designed specific primers, its The sequence is:

[0033] Dmrt1-F: 5'-ATGAACAAGGACAAGCAGAG-3';

[0034] Dmrt1-R: 5'-ATTGATGCCATCCACGTCGA-3'

[0035] PCR amplification was carried out. After the amplified fragments were verified by gel recovery, purification and sequencing, they were connected to the linearized Blunt E2 vector. The recombinant plasmids were transformed into E. coli DH5α competent cells. PCR amplification detection, and handed over to Ruibo Xingke Biotechnology (Beijing) Co., Ltd. for sequencing identification.

[0036] Amplification conditions are:

[0037] Pre-denaturation: 2min at 94°C; denaturation, annealing and extension: 30s at 94°C, 30s at 60°C, 1min 30s at 72°C, a total of 35 cycles; final extension: 5min at 72°C.

[0038] SEQ ID NO: 1 is that the amino acid sequence of Dmrt1 is:

[0039]

[0040] The recombinant plasmid Blunt E2-Dmrt1 was transformed into E. coli Transetta (DE3), spread on LB solid medium containing 0.1% kanamycin, and cultured overnight at 37°C by inversion. Then a single colony was picked for bacterial liquid PCR detection and identification. The PCR product was subjected to 1% agarose gel electrophoresis, and the size of the target band of the amplified product was observed under a gel imager, and the colony that was consistent with the expected fragment size was a recombinant strain.

[0041] 2. Induced Expression of Recombinant Strains

[0042] 2.1 Induction and expression of recombinant strains: Inoculate the recombinant strains on LB solid medium plates containing 0.1% kanamycin by streaking, and inoculate single colonies containing 0.1% kanamycin after inverted culture at 37°C for 14-16 hours. The LB liquid medium of kanamycin was placed at 37°C and cultured at 200 r/min for 12-16 h as the first-class seeds; the first-class seeds were inoculated into the medium containing 0.1% kanamycin at 1% inoculum. IPTG (0.5 mM) was added, and the cells were incubated at 37° C. and 200 rpm for 16 h, and the bacterial liquid was collected.

[0043] 2.2 Collection and detection of Dmrt1 recombinant protein: the bacterial solution was centrifuged at 4,000 g for 10 min at 4 °C, and the bacterial cells were collected, PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na) 2 HPO 4 , 1.8mMKH 2 PO 4 , pH 7.4) after washing 3 times, resuspended with bacterial lysate containing protease inhibitor, and lysed by sonication at 250W on ice for 25min. Centrifuge at 10,000 g for 15 min at 4°C and collect the pellet. Dmrt1 recombinant protein was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) (see figure 1 A).

[0044] The above-mentioned expression yield of the recombinant protein obtained from the Blunt E2 vector is low, so that the recombinant protein cannot be further analyzed and applied. It can be seen that the selected vector may not be suitable.

Example Embodiment

[0045] Example 2

[0046] 1. Construction of recombinant plasmid pET-30a-EGFP-Dmrt1 and recombinant strain

[0047] Extracting the RNA of the flounder testis tissue, and reverse transcribing it into cDNA, using PCR to amplify the entire coding region of the flounder Dmrt1, the encoded amino acid sequence is shown in SEQ ID NO: 1, and then using the designed specific primers, its The sequence is:

[0048] Dmrt1-F: 5'-AAGGCCATGGCTGATATGAACAAGGACAAGCAGAG-3';

[0049] Dmrt1-R: 5'-TGAATCGATACGCATATTGATGCCATCCACGTCGA-3'

[0050] PCR amplification was performed. After the amplified fragment was verified by gel recovery, purification and sequencing, it underwent homologous recombination with the linearized pET-30a-EGFP vector. The recombinant plasmid was transformed into E. coli DH5α competent. The cloned colonies were detected by PCR amplification of the bacterial solution, and were handed over to Ruibo Xingke Biotechnology (Beijing) Co., Ltd. for sequencing and identification.

[0051] Amplification conditions are:

[0052] Pre-denaturation: 2min at 94°C; denaturation, annealing and extension: 30s at 94°C, 30s at 60°C, 1min 30s at 72°C, a total of 35 cycles; final extension: 5min at 72°C.

[0053] SEQ ID NO: 1 is that the amino acid sequence of Dmrt1 is:

[0054]

[0055] The recombinant plasmid pET-30a-EGFP-Dmrt1 was transformed into E. coli Transetta (DE3), spread on LB solid medium containing 0.1% kanamycin, and cultured overnight at 37°C by inversion. Then a single colony was picked for bacterial liquid PCR detection and identification. The PCR product was subjected to 1% agarose gel electrophoresis, and the size of the target band of the amplified product was observed under a gel imager, and the colony that was consistent with the expected fragment size was a recombinant strain.

[0056] 2. Induction and expression of recombinant strains and purification of Dmrt1 recombinant protein

[0057] 2.1 Induction and expression of recombinant strains: Inoculate the recombinant strains on LB solid medium plates containing 0.1% kanamycin by streaking, and inoculate single colonies containing 0.1% kanamycin after inverted culture at 37°C for 14-16 hours. The LB liquid medium of anamycin was placed at 37°C and cultured at 200 rpm for 12-16 h as the first-class seeds; the first-class seeds were inoculated with 1% inoculum in the automatic induction compound culture containing 0.1% kanamycin In the base, placed at 37 ° C, 200 r/min for 16 h, and the bacterial liquid was collected.

[0058] 2.2 Collection and purification of Dmrt1 recombinant protein: The bacterial solution was centrifuged at 4,000g for 10 min at 4°C to collect the bacterial cells, washed with PBS for 3 times, resuspended with bacterial lysate containing protease inhibitors, and lysed by ultrasonic at 250W on ice for 25 min. Centrifuge at 10,000 g for 15 min at 4°C and collect the pellet.

[0059] Resuspend in 10 mL of equilibration buffer (5 mM imidazole, 0.5 mM NaCl, 8 M urea, 20 mM Tris-HCl, pH 7.9) per gram of protein pellet, vortex at room temperature After shaking for 10 min, centrifuge at 10,000 g for 20 min at 4°C to collect the supernatant. Load the supernatant onto the column (Ni 2+ column), rinsed with 15 column volumes of equilibration buffer to remove impurities. Elution was carried out with an appropriate amount of elution buffer (500 mM imidazole, 0.5 mM NaCl, 8 M urea, 20 mM Tris-HCl, pH 7.9), and the elution peaks were collected.

[0060] 3. Dialysis renaturation of Dmrt1 recombinant protein

[0061] The purified protein was placed in an MD34 (Mw50,000) dialysis bag, and soaked in a volume ratio of 1:100 of protein sample and dialysis buffer. The main components of the dialysis buffer were 50mM Tris, 24mM NaCl, 1mM KCl, 0.9mM L-glutathione reduction (GSH), 0.1 mM L-glutathione oxidation (GSSG) and different concentrations of imidazole (250 mM, 100 mM, 0 mM). The dialysis time for each gradient was 6 h, the last gradient was repeated twice, and the dialyzed proteins were stored at -80°C. The Dmrt1 recombinant protein after dialysis and the uninduced recombinant bacterial protein were analyzed by SDS-PAGE to determine the molecular weight ( figure 1 B, C), in line with the expected protein size. This indicated that the Dmrt1 recombinant protein existed in the form of insoluble inclusion bodies, and the recombinant protein with higher purity was obtained after purification. From this, the recombinant protein of Dmrt1 was obtained, protein length: 302 amino acids, type: amino acid, chain type: single chain, characteristic: predicted molecular weight of 33.0kDa, isoelectric point of 6.30, with conserved DM domain and Pfam domain .

Example Embodiment

[0062] Example 3 In vitro activity verification of Dmrt1 recombinant protein

[0063] 1. Tissue incubation of Dmrt1 recombinant protein

[0064] The recombinant and purified flounder Dmrt1 protein in Example 2 was diluted to 1000 μg/mL in the aforementioned PBS.

[0065] Take the gonadal tissues of four healthy wild-type male and female adult fish, wash them repeatedly with the aforementioned PBS containing 1% penicillin (10kU/mL)-streptomycin (10mg/mL) mixed antibiotics, and cut them into about 1.0mm 3 The small pieces were placed in a 24-well plate, pre-incubated with L15 medium containing 1% antibiotics at 25 °C for 6 h, followed by Dmrt1 recombinant protein (100 μg) and Lipofectamine containing recombinant and purified Dmrt1 in Example 2. TM The fresh medium of 3000 transfection reagent was incubated at 25°C for 24h, and the incubated samples were stored at -80°C for later use.

[0066] 2. WB and qPCR analysis

[0067] A part of the samples were extracted with total protein by radioimmunoprecipitation (RIPA), and after adding 5× loading buffer, they were bathed in boiling water for 8-10 minutes for WB detection. The remaining samples were extracted with total RNA by Trizol method, and the concentration and purity of RNA were detected by NanoDrop 2000 spectrophotometer and 1.5% agarose gel electrophoresis. The first-strand cDNA was synthesized using a reverse transcription kit for qPCR analysis of gene expression.

[0068] The results show that the recombinant protein can enter the gonadal tissue with the help of in vitro transfection reagents, see figure 2 A. After incubation with Dmrt1 recombinant protein, the expression of cyp19a gene in ovary and testis was significantly decreased (p<0.05), as shown in figure 2 shown in B. The results showed that the purified Dmrt1 recombinant protein could significantly inhibit the expression of cyp19a in gonads (p<0.05), and had biological activity.

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