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Recombinant large-tooth flounder interleukin-6 protein and preparation method thereof

A technology of interleukin and flounder, applied in the field of biogenetic engineering, can solve the problems of incapable of large-scale industrial production, complicated operation, high cost, etc., and achieve the effects of convenient identification and purification, simple operation, and reduced production cost

Inactive Publication Date: 2010-08-18
DALIAN FISHERIES UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the direct extraction of the protein from flounder is complex, costly, and low in yield, and cannot be produced on a large scale.

Method used

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  • Recombinant large-tooth flounder interleukin-6 protein and preparation method thereof
  • Recombinant large-tooth flounder interleukin-6 protein and preparation method thereof
  • Recombinant large-tooth flounder interleukin-6 protein and preparation method thereof

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Embodiment

[0027] a. The total RNA of fresh flounder liver was extracted with TRIzol Reagent, and the first-strand cDNA was synthesized by reverse transcription. Agarose gel electrophoresis analysis of flounder total RNA figure 1 As shown, in the figure 1: MakerDL2000; 2, 3: electrophoresis bands of total RNA of flounder.

[0028] b. According to the conserved sequence of the flounder interleukin 6 protein gene published in GenBank, a primer pair was designed for the coding sequence of the interleukin 6 protein, and PCR amplification was carried out with the above-mentioned first-strand cDNA. The nucleotide sequence of the primer pair was :

[0029] Upstream primer IL-6F: 5'-ca ggatccg ctccagtcgaatacgag-3';

[0030] Downstream primer IL-6R: 5'-cg ctcgag tgtcatttggtaagaggg-3'.

[0031] The RT-PCR reaction conditions were as follows: 94°C pre-denaturation for 5 minutes followed by 32 cycles of amplification at 94°C for 30 seconds, 64°C for 30 seconds, 72°C for 1 minute, and 72°C for...

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Abstract

The invention discloses a recombinant large-tooth flounder interleukin-6 protein and a preparation method thereof. The preparation method comprises the following steps: according to a DNA sequence of the large-tooth flounder interleukin-6 protein of paralichthys olivaceus, designing a primer and extracting the total RNA of the kidney fresh by a TRIzol Reagent method, carrying out RT-PCR augmentation, connecting a PCR product and a Pmd19T vector to form a recombinant plasmid, connecting with an expression vector Pet-32a(+) by double digestion, obtaining the recombinant plasmid and transforming the recombinant plasmid into escherichia coli BL21 (DE3); and realizing the soluble expression of the recombinant protein. As the expression process does not generate an inclusion body, the fusion protein for expressing the plasmid has histidine tags, thus being convenient to identification and purification of samples, avoiding the denaturation and renaturation of the samples, having simple operation, reducing the manufacturing cost, and more importantly, having no damage to a protein structure; and the invention has good purity of recombinant protein, high yield and difficult degradation, is applicable to industrial production and lays the foundation of studying the large-tooth flounder interleukin-6 protein of the paralichthys olivaceus.

Description

Technical field: [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a recombinant flounder interleukin-6 protein which is easy to operate, low in cost, high in purity and high in yield, and suitable for industrial production, and a preparation method thereof. Background technique: [0002] Flounder (Paralichthys olivaceus) is a rare marine fish in my country and one of the important economic fishes in marine culture in China. Flounder is large in size and contains high protein, low fat, rich vitamin B1, vitamin B2 and vitamin D and other nutrients. At present, there is a large demand in domestic and foreign markets, and the supply of products exceeds demand. However, in recent years, while the aquaculture area of ​​flounder has continued to expand, the disease of the cultured flounder has increased year by year due to the deterioration of seawater quality pollution, coupled with poor management and drug abuse, which h...

Claims

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Application Information

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IPC IPC(8): C07K14/54C12N15/24C12N15/70
Inventor 王秀利潘秋丽袁野刘洋张峰仇雪梅
Owner DALIAN FISHERIES UNIVERSITY
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