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Lateolabrax japonicus FGF6A, FGF6B and FGF18 recombinant protein as well as preparation method and application thereof

A technology of FGF6B and recombinant protein, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve research blanks and other problems

Active Publication Date: 2022-03-15
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The molecular mechanism of FGFs-FGFR pathway regulating the proliferation and differentiation of skeletal muscle cells is still in the exploratory stage, and the relevant research in fish is still blank

Method used

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  • Lateolabrax japonicus FGF6A, FGF6B and FGF18 recombinant protein as well as preparation method and application thereof
  • Lateolabrax japonicus FGF6A, FGF6B and FGF18 recombinant protein as well as preparation method and application thereof
  • Lateolabrax japonicus FGF6A, FGF6B and FGF18 recombinant protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of perch FGF6A, FGF6B and FGF18 expression vector pET-28a(+)-SUMO-FGF

[0037] Using perch muscle cDNA as a template, PCR was performed using primers FGF6A-F / R, FGF6B-F / R, FGF18-F / R (Shenggong, Shanghai) and reagent 2×Phanta Max Master Mix (Novizant, Nanjing) The DNA sequences of the open reading frames (ORF) of the perch FGF6A, FGF6B and FGF18 genes were amplified. It was ligated into pCE2 TA / Blunt Zero vector (Novazyme, Nanjing) by TOPO ligation to transfect DH5α Escherichia coli, a single colony was screened on ampicillin solid medium, and Sanger sequencing and NCBI BLAST were used to identify whether the sequence was correct. Using the above-mentioned monoclonal cultures of perch FGF6A, FGF6B and FGF18 as templates, PCR amplification was performed using primers PE-FGF-F / R and 2×Phanta Max Master Mix to obtain mature FGF6A, FGF6B and FGF18 with homology arms. The double-stranded DNA fragment corresponding to the peptide; meanwhile, the pET-28a(...

Embodiment 2

[0041] Example 2: Preparation of sea bass FGF6A, FGF6B and FGF18 Escherichia coli expression strains

[0042] The expression vector pET-28a(+)-SUMO-FGF was transfected into BL21(DE3) competent Escherichia coli (Ang Yu, Shanghai), and cultured with kanamycin-resistant LB solid medium at 37°C overnight to screen a single colony, namely The engineering strain pET-28a(+)-SUMO-FGF-BL21(DE3) was used to express the prokaryotic expression of FGF6A, FGF6B and FGF18 of perch. Pick a single colony and culture it overnight in 10 mL of kanamycin-resistant LB liquid medium at 37°C and 220 rpm, and then inoculate it in 100 mL of kanamycin-resistant LB liquid medium at a ratio of 1:100. Cultivate at 220 rpm until OD600 reaches 0.4-0.5, then add IPTG to make the final concentration 0.15mmol / L (take out 50mL of bacterial liquid as a control before induction), at 20℃, 120 rpm and culture for 12h, 4000× Centrifuge at g for 10 min to collect the bacteria, discard the supernatant, resuspend the b...

Embodiment 3

[0043] Example 3: Soluble expression and protein purification of sea bass FGF6A, FGF6B and FGF18 recombinant proteins

[0044] Inoculate in 500mL kanamycin-resistant LB liquid medium at a ratio of 1:100, culture at 37°C, 220 rpm until OD600 reaches 0.4-0.5, then add IPTG to make the final concentration 0.15mmol / L, 20 Centrifuge at 120 rpm for 12 hours, then centrifuge at 4000×g for 10 minutes to collect the bacteria, discard the supernatant, resuspend the bacteria in 50 mL of non-denaturing lysis buffer, add lysozyme at a final concentration of 1 mg / mL, and lyse on ice for 30 minutes, 60W Sonicate for 15 min, ultra-sonicate for 5 s and stop for 5 s, centrifuge at 12,000×g for 10 min to discard the precipitate, retain the supernatant, filter with a 0.45 μm or 0.22 μm filter membrane to remove insoluble particles, and use TED-Ni filler (Abbison, Shanghai) for His Tag affinity purification to obtain FGF6A, FGF6B and FGF18 recombinant proteins ( image 3 Middle sample 8-9), can b...

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Abstract

The invention provides a lateolabrax japonicus FGF6A, FGF6B and FGF18 recombinant protein as well as a preparation method and application thereof, the lateolabrax japonicus FGF6A, FGF6B and FGF18 recombinant protein comprises FGF6A, FGF6B and FGF18 mature peptide amino acid sequences and tag peptide fragments connected to N ends of the FGF6A, FGF6B and FGF18 mature peptide amino acid sequences, and the tag peptide fragments comprise group amino acid tag sequences and dissolution promoting tag sequences. The invention also provides a gene for coding the recombinant protein, a vector for expressing the recombinant protein and a recombinant engineering bacterium. The invention further provides a preparation method of the recombinant protein and application of the recombinant protein in breeding of lateolabrax japonicus. Soluble FGF6A, FGF6B and FGF18 recombinant proteins can be obtained through large-scale induced expression of prokaryotic bacteria, and the recombinant proteins have activity and are beneficial to subsequent industrial extraction and purification.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an Escherichia coli expression system for producing soluble and biologically active perch FGF (FGF6A, FGF6B and FGF18) recombinant proteins, a preparation method and its application in perch breeding. Background technique [0002] Lateolabrax maculatus, commonly known as sea bass, belongs to the order Perciformes, the family Serranidae, and the genus Lateolabrax. Its tender meat, delicious taste and high nutritional value are widely loved by consumers. Perch is a typical wide-temperature and wide-salt fish with strong resistance to stress and disease. It can make good use of artificial compound feed. It can grow well in ponds, factories, cages and other water bodies. It has a very broad farming prospects. In recent years, the annual output of perch farming in my country has exceeded 150,000 tons, ranking among the top three in marine fish production, and ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/50C12N15/12C12N15/62C12N15/70C12N1/21A61K38/18A61P21/00C12R1/19
CPCC07K14/50C12N15/70A61P21/00C07K2319/21C07K2319/95A61K38/00Y02A40/81
Inventor 李昀齐鑫温海深张凯强董夕梦张静茹王孝杰陈基伟
Owner OCEAN UNIV OF CHINA
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