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Eukaryotic expression vector containing mature peptide gene of epinephelus coioids gonadotropin hormone I, recombinant strain, recombinant protein and application thereof

A eukaryotic expression carrier and gonadotropin technology, applied in the field of genetic engineering, can solve the problems of restricting grouper breeding production, unstable effect, high cost, etc., and achieve the effect of low preparation cost, remarkable effect and great economic value

Active Publication Date: 2011-06-01
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The asynchrony of grouper gonad development and ejaculation and ovulation restricts the aquaculture production of grouper. At present, the existing oxytocic agents such as hCG or fish pituitary extract are high in cost and unstable in effect.

Method used

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  • Eukaryotic expression vector containing mature peptide gene of epinephelus coioids gonadotropin hormone I, recombinant strain, recombinant protein and application thereof
  • Eukaryotic expression vector containing mature peptide gene of epinephelus coioids gonadotropin hormone I, recombinant strain, recombinant protein and application thereof
  • Eukaryotic expression vector containing mature peptide gene of epinephelus coioids gonadotropin hormone I, recombinant strain, recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Synthesis of Gonadotropin I Single Subunit Gene GTHα, FSHβ Mature Peptide

[0055] According to the gonadotropin α (accession number: AY129308 / AY129309) gene sequence and FSHβ (accession number: AY553982 / AY679087) gene sequence registered in GeneBank, design specific primers: Pα1:5,-taccccaacattgacttacc -3, Pα2:5, -tcatatcttgtggaaatagcaggtgc-3,; P I β1:5, -gactgtcatctgaccagcat-3, P I β2:5, -ttaaaaggac agacag ctgg at-3, inversion of total RNA from the pituitary gland of the grouper The cDNA of the recorded product was used as a template, and the target fragments were amplified respectively. The reaction conditions were: pre-denaturation at 94°C, 4min; denaturation at 94°C, 30s; annealing cga at 50°C / FSHβ at 50°C, 30s; cycle; 72 ° C insulation, 10min. The electrophoresis identification chart of the PCR amplification product is shown in figure 1 , the size of the target fragment GTHα is 285bp, FSHβ is 300bp.

Embodiment 2

[0056] Example 2 Cloning vectors pPTZ 57R / T-GTHα and pPTZ 57R / T-FSHβ and recombinant strains pPTZ 57R / T-GTHα-DH5α and pPTZ 57R / T containing the mature peptide gene of gonadotropin I single subunit of the grouper - Construction of FSHβ-DH5α.

[0057] The target gene fragment obtained in Example 1 was isolated and purified, then mixed with the TA vector pPTZ 57R / T at a molar ratio of 3:1, and ligated overnight at 22°C (about 16h) using T4 ligase (ferminant company), and the ligated product was transformed Escherichia coli DH5α, positive clones were screened by Amp+ resistance and blue and white spots, and the plasmid was extracted by a plasmid extraction kit. The plasmid was verified by double enzyme digestion and sequencing, which proved that the mature peptide gene of the gonadotropin I single subunit of the oblique grouper Cloned into vectors, named pPTZ 57R / T-GTHα and pPTZ 57R / T-FSHβ respectively, and the recombinant strains obtained by transforming Escherichia coli DH5α wit...

Embodiment 3

[0058] Example 3 Construction of gene expression vectors pPICZαA-GTHαh, pPICZαA-FSHβh, pPICZαA-FSHβhm containing gonadotropin I single subunit mature peptide gene expression vectors of grouper

[0059] According to the gonadotropin α (accession number: AY129308 / AY129309) gene sequence and FSHβ (accession number: AY553982 / AY679087) gene sequence registered in GeneBank, design specific primers: Pα3: 5'-g gaattc taccccaacattgacttacc-3',Pα4:5'-tcc ccgcgg tta atgatgatgatgatgatgtatcttgtggaaat-3', where Gaattc is the EcoRI restriction site, and g is its protective base; ccgcgg is the SacII restriction site, and tcc is its protective base; tctaga is the Xba I restriction site, and gc is its protective base base; atgatgatgatgatgatg for 6 × his-tag; PIβ3: 5'-g gaattc gactgtcatctgaccagcat-3', PIβ4:5'-gg ggtacc ttaatgatgatgatgatgatgaaaggacagacag-3', PIβ5:5'-gc tctaga aaggacagacagctggat-3', where Gaattc is the EcoRI restriction site, and g is its protective base; ggtacc is th...

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Abstract

The invention discloses a cloning vector containing a mature peptide gene GTHalpha or FSHbeta of an epinephelus coioids gonadotropin hormone, an eukaryotic expression vector, a recombinant strain, a recombinant protein and application of the recombinant protein to the preparation of a fish oxytocic. Epinephelus coioids belongs to a rare marine fish and has high economic value. The recombinant protein for recombining the epinephelus coioids gonadotropin hormone I is produced by adopting a gene-engineering technology and can be used for preparing the fish oxytocic with low preparation cost and remarkable effect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a cloning vector, an expression vector, a recombinant bacterial strain, a recombinant protein and an application thereof containing a gonadotropin I mature peptide gene of a grouper. Background technique [0002] Gonadotropin hormone (GTH) is a glycoprotein of two subunits produced by the pituitary gland. Its target tissues are the testis and ovary. Its physiological function is mainly to increase the production and secretion of gonadal steroid hormones and promote gamete Generation, gonadal maturation and ejaculation ovulation. Gonadotropin hormone (GTH) is divided into two types, namely gonadotropin I (GTH I, also known as follicle-stimulating hormone (FSH)) and gonadotropin II (GTH II, also known as corpus luteum Genotropin (luteinizing hormone, LH)), gonadotropin I is a heterodimer composed of GTHα and FSHβ, and gonadotropin II is a heterodimer compose...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/16C12N15/79C12P21/02
Inventor 李文笙陈军
Owner SUN YAT SEN UNIV
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