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Hexameric prokaryotic expression vector of Cyprinus carpiovar Jian gonadotrophin-releasing hormone gene, and establishing method and application thereof

A gonadotropin, prokaryotic expression technology, applied in the application field of preparing anti-GnRH polyclonal antibodies

Inactive Publication Date: 2012-03-21
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of recombinant vectors that highly express the GnRH gene can efficiently obtain recombinant proteins to meet its application in fish artificial reproduction. At the same time, the prepared antibodies have not been reported in the existing research

Method used

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  • Hexameric prokaryotic expression vector of Cyprinus carpiovar Jian gonadotrophin-releasing hormone gene, and establishing method and application thereof
  • Hexameric prokaryotic expression vector of Cyprinus carpiovar Jian gonadotrophin-releasing hormone gene, and establishing method and application thereof
  • Hexameric prokaryotic expression vector of Cyprinus carpiovar Jian gonadotrophin-releasing hormone gene, and establishing method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Amplification and TA cloning of GnRH gene:

[0036] The amplification of GnRH gene and the strategy of TA cloning are as follows: figure 1 As shown, first search the full-length gene sequence of GnRH from GenBanK (GenBanK number: AY148223), and use Primer5.0 to design a pair of specific primers:

[0037] Primer 1: 5'-ACTGAGGGAAACTTGGACTGAT-3', see SeqNO.2;

[0038] Primer 2: 5'-CAAGGCAGAAATAGAAAGGAAG-3', see SeqNO.3;

[0039] The total RNA from the thalamus of Jian carp (taken from the experimental fish farm of Freshwater Fisheries Research Center) was extracted by RNA extraction kit, and then the first-strand cDNA was synthesized after purity identification, and PCR amplification was performed using the reverse transcription product as a template and the synthesized upstream and downstream primers , the reaction conditions were: 94°C pre-denaturation for 3 min, 94°C pre-denaturation for 30 s, 55°C annealing for 45 s, 72°C extension for 1 min, 30 cycles...

Embodiment 2

[0040] Embodiment 2: Construction of prokaryotic expression vector pMAL-GnRH:

[0041] After obtaining the correct GnRH sequence, go to the biological company to synthesize the tandem hexamer GnRH and GAP sequence, and use it and the prokaryotic expression plasmid pMAL-c2x respectively Eco RI, Hind Ⅲ Double enzyme digestion at 37°C, recover the GnRH fragment (including the tandem hexamer GnRH and GAP sequence) and the linearized pMAL plasmid with a gel recovery kit, design the ligation reaction system according to the instructions of T4 DNA ligase, and construct the expression plasmid pMAL-GnRH ( figure 2 ), and transformed into Escherichia coli JM109, positive clones were screened, 10 clones were randomly picked to extract plasmids, and the extracted plasmids were subjected to Eco RI and Hind Ⅲ Double enzyme digestion identification, the results of enzyme digestion showed that there was a band of about 400 bp after pMAL-GnRH digestion ( Figure 4 ), indicating that G...

Embodiment 3

[0042] Embodiment 3: Expression and identification of recombinant GnRH protein:

[0043] Transform pMAL-GnRH into Escherichia coli TB1, pick positive clones and inoculate them in LB liquid medium containing 50 μg / mL ampicillin, culture overnight at 37°C with shaking, take the culture solution and inoculate it in a fresh medium containing 50 μg / mL ampicillin Ampicillin-based LB liquid medium was induced with 0.5 and 1 mol / L IPTG respectively, and cultured with shaking at 37°C for 4 h, the bacteria were collected, resuspended in 100 μL SDS-PAGE loading buffer, centrifuged and added The loading buffer was boiled to break the cells, and the SDS-PAGE electrophoresis at 12% concentration was carried out according to "Molecular Cloning" and the reagent manual ( Figure 5 ) to identify the expression level of the protein. Depend on Figure 5 It can be seen that after induction by adding IPTG, a protein band with a molecular weight of about 42 kD appeared in the total protein extract...

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Abstract

The invention discloses a prokaryotic expression vector pMAL-GnRH for expressing polymeric gonadotrophin-releasing hormone efficiently. By an RT-PCR method, a target sequence GnRH gene with the length of about 400bp is amplified from the thalamencephalon of the Cyprinus carpiovar Jian and cloned into a T vector; after the correctness of the sequence is determined through the enzyme cutting identification and the sequence detecting analysis, a GnRH serial hexamer is manually synthesized and is coupled with GAP protein and the segment is cloned into an expression vector pMAL-c2x to establish recombinant expression plasmid pMAL-GnRH; and the expression product has an immunogenicity and can stimulate a body to generate an immune response.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and in particular relates to a prokaryotic expression vector for highly expressing Jiancyp gonadotropin (GnRH) and its application in prokaryotic expression of GnRH protein and preparation of anti-GnRH polyclonal antibody. [0002] Background technique [0003] Gonadotropin-releasing hormone (GnRH) precursor protein is composed of signal peptide, GnRH decapeptide, and gonadotropin-releasing hormone-related peptide (GnRHassociated peptide, GAP). Active GnRH decapeptide. GnRH plays a very important role in regulating the reproductive development of vertebrates, and is the key information molecule of the hypothalamic-pituitary-gonadal (HPG) axis, which stimulates the release of gonadotropin (GtH) from the anterior pituitary. ), which plays an important role in the development of gonads and the maintenance of reproductive function in vertebrates. There is no definite statement about th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C07K7/23
Inventor 丁炜东曹哲明曹丽萍邴旭文
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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