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Expression system of tilapia neuropeptide Y recombinant protein

A technology of recombinant protein and expression system, which is applied in the field of expression system of tilapia neuropeptide Y recombinant protein, to achieve the effect of promoting fish growth, low cost and stable source

Inactive Publication Date: 2010-09-08
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at present, there is no genetically engineered product feed additive that can effectively promote the growth of tilapia in artificial culture.

Method used

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  • Expression system of tilapia neuropeptide Y recombinant protein
  • Expression system of tilapia neuropeptide Y recombinant protein
  • Expression system of tilapia neuropeptide Y recombinant protein

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Cloning of Tilapia Neuropeptide Y Gene Fragment

[0032] Total RNA was extracted from the hypothalamus of Gifu tilapia ( figure 1 ), reverse-transcribed into cDNA with AP primers. Using cDNA as a template, a pair of primers were designed according to the published sequence of Oreochromis sp.YC-2004 (red tilapia) neuropeptide Y gene (GenBank: AY779047.1). Primers: the upstream primer is shown in SEQ ID NO: 4, The downstream AUAP clones the gene sequence from the start codon of the neuropeptide Y gene to the PolyA tail. The PCR reaction conditions are: 95°C pre-denaturation for 3 minutes; the following is 35 cycles, 95°C denaturation for 15 seconds, 53°C annealing for 15 seconds, 72°C Extension for 60 seconds; final extension for 10 minutes at 72°C.

[0033] The electrophoresis identification chart of the PCR amplification product is shown in figure 2 . From figure 2 It can be seen that a sequence of about 630 bp was amplified by the first PCR, and the se...

Embodiment 2

[0034] Example 2 Gene synthesis of neuropeptide Y gene of Gifu tilapia with enzyme-cut linker

[0035]According to the above cloned nerve body Y gene sequence and the recognition sequences of restriction endonucleases XhoI and XbaI, design and synthesize two pairs of specific primers:

[0036] The upstream primer A is shown in SEQ ID NO: 1, with a total of 43 bp, which is added with an XhoI restriction recognition site and a signal peptide coding sequence behind it before the mature peptide sequence of the NPY gene.

[0037] The downstream primer B is shown as SEQ ID NO: 2, with a total of 45 bp, which is added with 6×His tag, stop codon and enzyme recognition site after the mature peptide sequence of NPY gene.

[0038] The downstream primer C is shown in SEQ ID NO:3. Use the carrier T-TiNPY obtained in Example 1 as a template to carry out a PCR reaction. The PCR reaction conditions are: 95°C pre-denaturation for 3 minutes; the following is 35 cycles, 95°C denaturation for 15...

Embodiment 3

[0040] Example 3 Construction of Pichia pastoris expression vectors D and E containing the neuropeptide Y gene of Gifu tilapia

[0041] 1 and 2PCR products were double-digested with restriction endonucleases XhoI and XbaI, and the products were digested with Gel Extraction Kit recovery, agarose gel electrophoresis, separation and purification of about 160bp and about 140 tilapia neuropeptide Y gene fragments; vector plasmid pPICZ A was cut with restriction endonucleases Xba I and Xho I to isolate and purify a large fragment of 3.6 kb, mixed with two neuropeptide Y gene fragments at a ratio of 1:3, and ligated with NEB T4 ligase at 16°C for 16 hours Transform Escherichia coli DH5a into Escherichia coli DH5a using the standard calcium chloride transformation method, use a low-salt LB plate to screen transformants with Zeocin resistance, and extract the plasmid ( Figure 5 ), obtained a recombinant plasmid with a size of about 3.8kb, by using 5-AOX as primer sequencing, the re...

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Abstract

The invention discloses an expression system of tilapia neuropeptide Y recombinant protein. Tilapia neuropeptide Y gene with a digestion joint is cloned into a pichia expression vector to obtain a recombinant pichia expression vector; and then the recombinant pichia expression vector is converted into a pichia competent cell to obtain a pichia pastoris recombinant strain, and the recombinant strain expresses the tilapia neuropeptide Y recombinant protein. The tilapia neuropeptide Y recombinant protein expression system can be used for obtaining the tilapia neuropeptide Y recombinant protein with stable source and low cost, and the recombinant protein can be used for preparing fry growth promoting agent or additive, and can effectively promote the growth of fishes.

Description

technical field [0001] The invention relates to the field of tilapia neuropeptide Y protein, in particular to an expression system of tilapia neuropeptide Y recombinant protein. Background technique [0002] Neuropeptide Y (NPY) is a 36-amino acid single-chain polypeptide first extracted from pig brain by Tatemoto in 1982. Its open reading frame is 300 bp in length and encodes 99 amino acids, including a signal peptide of 28 amino acids. , a 36 amino acid mature peptide and a peptide tail of unknown function, the 36 amino acid mature peptide is the true active neuropeptide Y. NPY is an endogenous orexigenic factor, which belongs to the pancreatic polypeptide family and has high homology with two enteroendocrine peptides (pancreatic peptide and peptide YY). NPY has two mutually antiparallel helical regions, a proline-rich helix and a Helix, the two helix regions have the characteristics of amphoteric ionization, and the stable tertiary structure is maintained by hydrophobi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N1/19C12N15/12C07K14/435C12P21/02A61K38/17A61P43/00A23K1/16C12R1/84
Inventor 李文笙王光中
Owner SUN YAT SEN UNIV
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