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Artificial porcine epidermal growth factor eukaryotic expression vector and preparation method and brewers yeast engineering bacteria thereof

A technology of epidermal growth factor and eukaryotic expression vector, which is applied in the field of genetic engineering, can solve the problems of high cost, inability to use directly, difficult feed promotion, etc., and achieve the effects of low production cost, reduced production cycle, and reduced investment

Inactive Publication Date: 2013-04-03
SHENZHEN PREMIXINVE NUTRITION CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of porcine epidermal growth factor disclosed in Chinese patent 200610114038.7 is also induced by methanol, so although the expression product contains porcine epidermal growth factor, it cannot be directly used as feed, and it needs to be purified before it can be used
Expression of porcine epidermal growth factor in Pichia pastoris is difficult to promote as feed due to the high cost of methanol removal and purification

Method used

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  • Artificial porcine epidermal growth factor eukaryotic expression vector and preparation method and brewers yeast engineering bacteria thereof
  • Artificial porcine epidermal growth factor eukaryotic expression vector and preparation method and brewers yeast engineering bacteria thereof
  • Artificial porcine epidermal growth factor eukaryotic expression vector and preparation method and brewers yeast engineering bacteria thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1: Construction of Saccharomyces cerevisiae secretory expression vector pYES2-Mfa-(S)pEGF

[0023] (1) Artificial synthesis of porcine epidermal growth factor gene

[0024] According to the amino acid sequence of porcine epidermal growth factor and the preference of Saccharomyces cerevisiae to codons, the Saccharomyces cerevisiae-preferred artificial porcine epidermal growth factor (S) pEGF gene was artificially designed, and its nucleotide sequence is as follows:

[0025] AAT TCT TAT TCT GAA TGT CCA CCA TCT CAT GAT GGT TAT TGT TTG CAT GGTGGT GTT TGT ATG TAT ATT GAA GCT GTT GAT TCT TAT GCT TGT AAT TGT GTT TTTGGT TAT GTT GGT GAA AGA TGT CAA CAT AGA GAT TTG AAA TGG TGG GAA TTG AGATAA (shown in SEQ ID NO. 1).

[0026] Compared with the codon of the natural porcine epidermal growth factor gene, the artificial porcine epidermal growth factor gene codon has 37 bases changed, and its amino acid sequence has 100% homology with the original gene amino acid sequence.

...

Embodiment 2

[0050] Embodiment 2. Preparation and transformation of engineering bacteria of artificial porcine epidermal growth factor (S) pEGF

[0051] (1) Preparation of commonly used solutions and medium for Saccharomyces cerevisiae expression system

[0052] YPD medium: 20g of peptone, 10g of yeast extract, 20g of glucose, (if plate medium, add 20g of agar powder), dissolve in 800mL of water, dilute to 1L, autoclave for 20min.

[0053] SC-U medium: 6.70g yeast nitrogen-free extract, 0.15g compound amino acid, (if making SC-U selective plate medium, add 20g agar powder), add 900ml deionized water, autoclave for 20min, cool Add 100ml of bacteria-filtered 20% glucose solution at 50°C, mix well, and store at 4°C for later use.

[0054] SC-U induction medium: 6.70g yeast nitrogen-free extract, 0.15g compound amino acid, add 800ml deionized water, autoclave for 20min, add 100ml filtered 20% glucose solution and 100ml filtered Bacterized 20% galactose solution, mix well, and store at 4°C fo...

Embodiment 3

[0067] Example 3. Shake flask culture, induced expression and identification of Saccharomyces cerevisiae engineered bacteria containing artificial porcine epidermal growth factor eukaryotic expression vector pYES2-MFα-(S)pEGF

[0068] a. Pick a single colony transformant (Saccharomyces cerevisiae engineered strain containing artificial porcine epidermal growth factor eukaryotic expression vector pYES2-MFα-(S)pEGF) in 15ml of SC-U medium (containing 2% cotton by weight Subsugar), 30 ℃ shaking culture overnight;

[0069] b. Measure the OD value of the above culture solution, inoculate an appropriate amount of the culture solution into 50ml of SC-U induction medium (containing 2% raffinose by weight), and culture overnight to make the OD value 0.4;

[0070] c. Centrifuge at 1500g for 5min at 4°C to collect the bacteria;

[0071] d. Use 1-2ml of SC-U induction medium (containing 2% raffinose by weight) to suspend the bacteria, and re-inoculate in 50ml of SC-U induction medium (co...

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Abstract

The invention discloses an artificial porcine epidermal growth factor eukaryotic expression vector and a preparation method and brewers yeast engineering bacteria thereof. A porcine epidermal growth factor gene is artificially synthesized according to the preference of brewers yeast to a porcine epidermal growth factor codon, and is shown as SEQ ID NO. 1 in a sequence table. The invention further provides a brewers yeast expression vector containing the gene and engineering bacteria with secretory expression of the gene, and further provides a method for obtaining the engineering bacteria. The artificial porcine epidermal growth factor gene has preference to the brewers yeast; the engineering bacteria of the gene have a brewers yeast signal peptide which is favorable for the secretory expression of the porcine epidermal growth factor and is induced by raffinose which can be used as feed instead of methanol during expressing the porcine epidermal growth factor; and the engineering bacteria and the expressed product can be directly used as feed without being purified and extracted, so that the cost and growth period of the porcine epidermal growth factor as feed are greatly reduced, and the application of the porcine epidermal growth factor in the feed industry becomes possible.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an artificial porcine epidermal growth factor eukaryotic expression vector, a preparation method thereof, and a Saccharomyces cerevisiae engineering bacterium. Background technique: [0002] Porcine epidermal growth factor (porcine epidermal growth factor, pEGF) is a growth factor with relatively high content in porcine colostrum. It is a powerful cell mitogen, which can stimulate cell division, proliferation and differentiation, promote ion exchange and Biological properties such as extracellular matrix synthesis, which have a very important impact on the development of the gastrointestinal tract of weaned piglets. Due to the sudden interruption of milk-derived epidermal growth factor due to weaning, the digestive system of piglets is not yet fully developed, and the resistance is weak. The feed conversion rate is greatly reduced, resulting in piglets p...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/81C12N1/19C12N15/66C12R1/865
Inventor 郭春华张兴友陈友慷周琳
Owner SHENZHEN PREMIXINVE NUTRITION CO LTD
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