Preparation method of recombinant fugu rubripe interferon gamma protein

A technology of red fin puffer and interferon, which is applied in the field of preparation of recombinant red fin puffer interferon gamma protein, can solve the problems of large-scale industrial production, complex protein operation, high cost, etc., and avoid denaturation and renaturation , The protein structure is not damaged, and the production cost is reduced

Inactive Publication Date: 2014-03-26
DALIAN OCEAN UNIV
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Problems solved by technology

However, the operation of directly extracting the protein from pufferfish is complicated, the cost is high, and the yield is low, so it cannot be produced on a large scale.

Method used

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  • Preparation method of recombinant fugu rubripe interferon gamma protein
  • Preparation method of recombinant fugu rubripe interferon gamma protein
  • Preparation method of recombinant fugu rubripe interferon gamma protein

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Experimental program
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Embodiment

[0026] a. Total RNA was extracted from the kidney of fresh puffer puffer with TRIzol Reagent, and the first-strand cDNA was synthesized by reverse transcription. The agarose gel electrophoresis analysis picture of the total RNA of puffer puffer figure 1 As shown, in the figure 1: Maker DL2000; 2, 3: total RNA electrophoresis bands of redfin puffer;

[0027] b. Carry out PCR amplification with the above-mentioned first-strand cDNA, and use PCR technology to obtain the target gene.

[0028] The nucleotide sequence of the primer pair is:

[0029] Upstream primer: 5'-CATG CCATGG GCTCCTACATCCCAGTAGAAATG-3'

[0030] Downstream primer: 5'-CCG CTCGAG ATGACCTCGAATCGACATCT-3'

[0031] The RT-PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, renaturation at 58°C for 30 seconds, extension at 72°C for 30 seconds, and 30 cycles of amplification at 72°C for 7 minutes and storage at 4 ℃, the primer pair was synthesiz...

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Abstract

The invention discloses a preparation method of a recombinant fugu rubripe interferon gamma protein. The method has advantages of simple operation, low cost, high purity of the recombinant protein and high yield, and is suitable for industrial production. The preparation method comprises steps of: extracting the total RNA of fresh liver of fugu rubripes and performing inverse transcription to synthesize cDNA of a first strand; performing RT-PCR amplification by adopting the cDNA of the first strand as a template, with the upstream primer being N[co] I:5'-CATGCCATGGGCTCCTACATCCCAGTAGAAATG-3', and with the downstream primer being X[ho] I:5'-CCGCTCGAGATGACCTCGAATCGACATCT-3'; establishing a clone vector, expressing the vector, transferring into an expression bacterial, and cultivating the bacterial strain to obtain strain; subjecting the collected strain to enlarging cultivation, inducing with IPTG and collecting the bacterial strain; and smashing the bacterial strain, lysing, purifying, and collecting purified liquid.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a preparation method of recombinant redfin pufferfish interferon gamma protein which is easy to operate, low in cost, high in purity and high in yield, and suitable for industrial production. Background technique [0002] Redfin Pufferfish ( Fugu rubripes ) is mainly distributed in the Yellow Sea, Bohai Sea and East China Sea in my country. It is a carnivorous fish at the bottom of the sea. Its meat is delicious and has high edible value. The biological characteristics of activity and high specificity have potential value in medical development and have broad prospects in clinical practice. Interferon (IFN) is a kind of protein with broad-spectrum antiviral activity secreted by recipient cells when the body is infected by viruses, or induced by nucleic acid, bacterial endotoxin, cytokinin, etc. glycoprotein). Studies have shown that IFN makes cells pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/23C12N15/70C07K14/57
Inventor 王秀利马普刘洋姜志强仇雪梅
Owner DALIAN OCEAN UNIV
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