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Transgenic salmon calcitonin gene yeast as well as preparation method and uses thereof

A salmon calcitonin and transgenic technology, which is applied in the fields of molecular biology technology and bioengineering, can solve the problems of incapability of oral administration, low expression amount and high cost, and achieve oral administration, high expression amount and lower production cost. Effect

Inactive Publication Date: 2008-05-21
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The invention provides a salmon calcitonin gene-transferred yeast and its preparation method and application, which can solve the problems of low expression level, need for purification, high cost and inability to orally administer salmon calcitonin in the expression system at present

Method used

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  • Transgenic salmon calcitonin gene yeast as well as preparation method and uses thereof
  • Transgenic salmon calcitonin gene yeast as well as preparation method and uses thereof
  • Transgenic salmon calcitonin gene yeast as well as preparation method and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0063] Example 1: Preparation of salmon calcitonin gene-transferred yeast:

[0064] 1. Preparation of salmon calcitonin gene:

[0065] The DNA sequence of the gene was designed by using the preferred codons of Saccharomyces cerevisiae, and the two partial single-strand sCT1 and sCT2 of the salmon calcitonin gene were synthesized by using a DNA synthesizer. Take 80 pmol each of the above single-stranded DNA fragments sCT1 and sCT2, mix them in a final volume of 80 μl of annealing buffer, put them in a water bath at 90°C for 5 minutes, and slowly cool down to room temperature. Add 5 mol / L NaCl to a final concentration of 300 mmol / L and 3 times the volume of ethanol, and place at -20°C overnight; centrifuge to remove the supernatant, and drain to remove residual ethanol. Precipitated DNA was dissolved in 1× buffer of Klenow enzyme reaction solution, containing dNTPs (0.5mmol / L final concentration) and 5U Klenow enzyme, bathed in water at 37°C for about 1 hour, and double-strande...

Embodiment 2

[0087] Example 2: Immunofluorescence assay of salmon calcitonin expressed on the surface of Saccharomyces cerevisiae:

[0088] 1. The cultivation steps before the fluorescent labeling of the transgenic yeast are as described in step 5 in Example 1.

[0089] 2. The steps for fluorescently labeling transgenic yeast are as follows:

[0090] (1) The cultures obtained according to the above time points were centrifuged at 3000-5000 g for 5-10 minutes at 4° C. in a refrigerated centrifuge.

[0091] (2) Wash the culture once in 1×PBS.

[0092] (3) The pellet was resuspended in 250 μl 1×PBS (containing 1 mg / ml BSA and 1 μg antibody).

[0093] (4) Let stand on ice for 30 minutes, shake gently to mix.

[0094] (5) Centrifuge at 3000-5000g for 5-10 minutes in a refrigerated centrifuge at 4°C.

[0095] (6) Wash the pellet again with 1×PBS.

[0096] (7) Resuspend the cells yAGA2-sCT in 1×PBS (containing 1 mg / ml BSA and 1 μg fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit Ig...

Embodiment 3

[0102] Example 3: Flow cytometric determination of salmon calcitonin expressed on the surface of Saccharomyces cerevisiae:

[0103] 1. The cultivation steps before the fluorescent labeling of the transgenic yeast are as in step 5 in Example 1.

[0104] 2. The step of fluorescently labeling the transgenic yeast is as in step 2 in Example 2.

[0105] 3. Read the parameters of the fluorescently labeled cells in the flow cytometer. In Figure 6, the abscissa represents the fluorescence reading, and the ordinate represents the number of cells. Each value in the graph represents the number of cells at that fluorescence reading. In the double peaks in the figure below, the first peak represents the yeast background, and the second peak represents the fluorescently labeled cell population. Flow cytometry can infer the number of expressing cells by automatically calculating the area under the curve. It was observed that with the increase of induction time, the number of cells expres...

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Abstract

The invention discloses transgenic salmon calcitonin yeast, a preparation method and the application. The salmon calcitonin is expressed in a eukaryotic system of Saccharomyces cerevisiae. The fermentation technology of Saccharomyces cerevisiae is mature with a higher expression and the Saccharomyces cerevisiae can be eaten directly; the obtained transgenic salmon calcitonin Saccharomyces cerevisiae can be taken orally without purification. The invention reduces the production cost and realizes oral administration of calcitonin.

Description

technical field [0001] The invention belongs to the field of molecular biology technology and bioengineering technology, and specifically relates to a salmon calcitonin gene-transferred yeast and its preparation method and application. Background technique [0002] Calcitonin (CT) is a polypeptide hormone composed of 32 amino acid residues, which is secreted by the posterior parotid body of vertebrates and thyroid C cells of mammals. Since the first discovery of calcitonin by Copp et al. in 1961, extensive studies have been conducted on its molecular structure and mechanism of action. It was found that calcitonin cooperates with thyroxine (PTH) in the human body to regulate the metabolism of calcium and phosphorus, and can inhibit the primary bone resorption and secondary bone resorption of bone. Calcitonin from different sources has different amino acid sequences. Calcitonin from different organisms has activity on humans. sCT for short) has the highest activity, which is...

Claims

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Application Information

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IPC IPC(8): C12N1/19C07H21/04A61K36/064A61P19/00A61P19/10
Inventor 张学成孙平楠臧晓南陈云松
Owner OCEAN UNIV OF CHINA
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