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Scatophagus argus germ cell maturation promoting gene IGF3 and application thereof

A germ cell and gene technology, applied in the field of genetic engineering, can solve the problems of restricting large-scale seedling cultivation and artificial ripening effect is not obvious

Active Publication Date: 2021-04-09
GUANGDONG OCEAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2015, the team where the applicant belongs made a breakthrough in the fully artificial breeding technology of money fish. However, in actual production, it was found that money fish is the same as other seawater fish. The effect of traditional artificial ripening is not obvious, which greatly limits the cultivation of large-scale seedlings.

Method used

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  • Scatophagus argus germ cell maturation promoting gene IGF3 and application thereof
  • Scatophagus argus germ cell maturation promoting gene IGF3 and application thereof
  • Scatophagus argus germ cell maturation promoting gene IGF3 and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] The preparation of embodiment 1 IGF3 recombinant protein

[0029] 1. Extraction of money fish RNA

[0030] Whole healthy goldfish was taken, anesthetized in an ice bath for about 2 minutes, the fish was killed and sampled, ovarian tissue was separated, and the total RNA of the ovarian tissue was extracted by Trizol reagent.

[0031] 2. Cloning of IGF3 gene

[0032] Synthesis of first-strand cDNA:

[0033] Using TaKaRa's PrimeScript TM RT reagent Kit with gDNA Eraser (PerfectReal Time) kit, firstly remove the genomic DNA in the total RNA of money fish ovary tissue, then reverse transcribe the total RNA to obtain the first-strand single-stranded cDNA, and store it in the -20°C refrigerator for later use.

[0034] IGF3 gene cloning using cDNA as template:

[0035] According to the relevant sequence alignment and structural analysis results in bioinformatics, 29 codes were removed from the cDNA sequence of the gene IGF3 (as shown in SEQ ID NO: 1, and its encoded protei...

Embodiment 2

[0050] Example 2 Mass Spectrometry Inspection of IGF3 Recombinant Protein

[0051] The first is the reductive alkylation of the protein, adding a final concentration of 10mM dithiothreitol (DTT) to reduce the IGF3 recombinant protein, then adding a final concentration of 55mM iodoacetamide (IAM), and finally adding 1μg of Trypsin enzyme, and enzymolysis overnight for 8h~ 16h. The polypeptide produced by enzymatic hydrolysis was desalted with a C18 column, and the desalted polypeptide was drained and dissolved with 15 μL Loading Buffer (0.1% formic acid, 3% acetonitrile). The peptides were analyzed by LC-MS / MS (ekspertTMnanoLC; AB Sciex TripleTOF 5600-plus) instrument, and then the results were evaluated. After LC-MS / MS off-board, submit the raw off-board data directly to AB SCIEX Triple TOF TM The database search was performed in the Proteinpilot software connected to the 5600plus mass spectrometer.

[0052] Mass spectrometry results (eg Figure 4 Shown) the sequence show...

Embodiment 3

[0054] Example 3 IGF3 recombinant protein regulates the expression of germ cell maturation regulatory genes

[0055] The money fish purchased from the Zhanjiang market was used as the research object, and the ovarian tissue specifically expressing the gene IGF3 was used as the material, and the IGF3 recombinant protein was used to incubate the ovarian fragments in vitro, and the IGF3 recombinant protein was used to study the relative germ cell maturation regulation genes cdc2 and cyclinB at the mRNA transcription level The regulation of expression.

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Abstract

The invention discloses a scatophagus argus germ cell maturation promoting gene IGF3. The nucleotide sequence of the scatophagus argus germ cell maturation promoting gene IGF3 is shown as SEQ IDNO: 3. The invention also discloses a protein for encoding the gene IGF3, cDNA of the scatophagus argus germ cell maturation promoting gene IGF3, and a protein for encoding the cDNA of the gene IGF3. The amino acid sequence of the protein for encoding the gene IGF3 is shown as SEQ IDNO: 4, the nucleotide sequence of the cDNA of the scatophagus argus germ cell maturation promoting gene IGF3 is shown as SEQ IDNO: 1, and the amino acid sequence of the protein for encoding the cDNA of the gene IGF3 is as shown in SEQ IDNO: 2. The invention also discloses an expression vector comprising the gene IGF3, a scatophagus argus IGF3 recombinant protein and a preparation method thereof, and an application of the gene IGF3, the protein for encoding the gene IGF3 or the recombinant protein in preparation of a medicine having a maturation promoting effect on scatophagus argus oocytes, and the developed IGF3 recombinant protein is expected to be applied to research and development of a novel fish maturation and spawning induction preparation.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene IGF3 for promoting germ cell maturation of money fish and its application. [0002] technical background [0003] Reproduction and growth are the most basic characteristics of living things; they are closely related but distinct from each other. Insulin-like growth factors (IGFs) are one of the key factors regulating the growth and reproductive axis. IGFs are composed of 3 ligands (IGF1, IGF2 and the newly discovered IGF3), 2 receptors (IGF1R, IGF2R) and 6 IGF-binding proteins (IGFBP). In fish, IGF1 and IGF2 mainly play the physiological function of promoting growth, but since the discovery of specifically expressed IGF3 in fish ovary, the role of IGFs in fish gonad development has received extensive attention. [0004] IGF3 is a fish-specific IGF type. It was first cloned and identified in zebrafish and tilapia, and it was found that IGF3 was spec...

Claims

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Application Information

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IPC IPC(8): C07K14/65C12N15/16C12N15/70A61K38/30A61K48/00A61P15/08C12R1/19
CPCC07K14/65C12N15/70A61K38/30A61K48/005A61P15/08
Inventor 陈华谱李智渊李水生张勇江东能田昌绪蒋谋炎朱春华李广丽
Owner GUANGDONG OCEAN UNIVERSITY
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