Turtle collagen gene functional fragment as well as recombinant protein and application thereof

A technology of collagen and gene function, applied in the field of genetic engineering, can solve the problem that there is no soft-shelled turtle collagen gene and its recombinant protein, and achieve good antioxidant activity, low cost, and short cycle

Active Publication Date: 2015-06-24
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is no report of soft-shelled turtle collagen gene and its recombinant protein

Method used

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  • Turtle collagen gene functional fragment as well as recombinant protein and application thereof
  • Turtle collagen gene functional fragment as well as recombinant protein and application thereof
  • Turtle collagen gene functional fragment as well as recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The present invention clones the soft-shelled turtle collagen gene functional fragment from the soft-shelled turtle, which has the nucleotide sequence shown in the sequence table SEQ ID NO.1, and expresses the gene to obtain the recombinant protein shown in the sequence table SEQ ID NO.2, The recombinant protein is the functional peptide of recombinant soft-shelled turtle collagen, which contains 27 unique Gly-X-Y repeat domains of collagen and has a molecular weight of 38kDa.

[0038] The preparation method of the above-mentioned recombinant soft-shelled turtle collagen functional peptide, the specific steps are as follows:

[0039] 1. Cloning of functional fragment cDNA of soft-shelled turtle collagen

[0040] 1.1 Extraction of soft-shelled turtle total RNA

[0041] The RNA extraction kit Trizol LS (purchased from Yingjun Company) was used to extract the total RNA of soft-shelled turtle according to the instructions, and the extracted RNA was stored at -80°C.

[004...

Embodiment 2

[0098] Determination of the antioxidant capacity of the recombinant soft-shelled turtle collagen functional peptide described in Example 1, the specific steps are as follows:

[0099] Refer to the method reported by Hu Wenting et al. (Hu Wenting, Sun Mi, Wang Yuejun. Separation, purification and properties of antioxidant peptides from Chlamys farreri [J]. Ocean and Limnology, 2006, 37(1): 14-19. ). A Fenton reaction system was used. The principle is: ferrous ions catalyze hydrogen peroxide to produce hydroxyl radicals, and the hydroxyl radicals produced by this reaction can fade the color of saffron. Antioxidants can scavenge hydroxyl radicals and inhibit the fading of saffron red. The specific operation is: take 4 test tubes, numbered 1, 2, 3, 4. Among them, No. 1 test tube was sequentially added with 0.025mol / L phosphate buffer 1mL, 40μg / mL saffron 1mL, 1mg / mL recombinant soft-shelled turtle collagen functional peptide 0.5mL, 3wt% hydrogen peroxide 1mL, 0.945 mmol / LEDTA-...

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Abstract

The invention discloses a collagen gene functional fragment cloned from a turtle and shown in sequence SEQ ID NO.1 in a sequence table and a recombinant protein obtained by the expression of the clone gene functional fragment and shown in sequence SEQ ID NO.2 in the sequence table, wherein the recombinant protein is a recombinant turtle collagen gene functional peptide fragment. The whole length of the recombinant turtle collagen gene functional peptide fragment includes 252 amino acids, 27 specific Gly-X-Y repeating fields of collagen are contained and the molecular weight is 38kDa. The preparation method comprises the steps of constructing a colibacillus genetic engineering bacterium for expressing the recombinant turtle collagen gene functional peptide fragment; culturing the colibacillus genetic engineering bacterium; inducing and expressing the recombinant turtle collagen gene functional peptide fragment; performing purification renaturation on the recombinant turtle collagen gene functional peptide fragment. The experiment shows that the recombinant turtle collagen gene functional peptide fragment prepared by the method provided by the invention has a high antioxidant activity and also has a value of application as an antioxidant.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to functional fragments of soft-shelled turtle collagen genes, recombinant proteins and applications thereof. Background technique [0002] Collagen is an extracellular structural protein mainly found in tendons, ligaments, cartilage, skin and connective tissues of humans and animals. Collagen is rich in amino acids such as glycine, proline, and hydroxyproline needed by the human body. The polypeptide products obtained by its hydrolysis not only have many important physiologically active functions but also have good digestion and absorption characteristics. This has made collagen a research hotspot in recent years, and it has also been widely used in medicine, food, beauty and biomaterials. [0003] The traditional main method of preparing collagen is to extract it from the skin, placenta and other tissues of animals such as cattle and pigs, which has many u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/78C12N15/70
Inventor 李登峰徐然刘联国
Owner NINGBO UNIV
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