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Method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds

A technology of epidermal growth factor and human serum albumin, applied in the direction of serum albumin, epidermal growth factor, genetic engineering, etc., can solve the problem of low expression and achieve the effect of good reproducibility, simple operation and low cost

Pending Publication Date: 2020-08-04
WUHAN HEALTHGEN BIOTECHNOLOGY CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The transgenic plant expression platform has the advantages of being safe, cheap, and easy to expand. In recent years, more and more recombinant drug proteins have been highly expressed in transgenic plants and have reached the requirements of industrial production. Current reports show that hEGF can be expressed in tomato leaves, potato Tubers, algae and tobacco are all expressed, but the expression level is generally low

Method used

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  • Method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds
  • Method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds
  • Method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of OsrHSA-EGF transgenic rice seeds

[0041] In this example, the rice-specific promoter Gt13a and its signal peptide are selected to mediate the expression of recombinant human serum albumin-epidermal growth factor gene in rice endosperm cells, specifically refer to the method in the publication number CN100540667 to construct the method of the present invention Rice specifically expresses recombinant human serum albumin-epidermal growth factor carrier and screens genetically engineered rice plants, and replaces said recombinant human serum albumin with the recombinant human serum albumin-epidermal growth factor of the present invention. use as figure 1 The indicated plasmid pOsPMP767 was used to construct the rice endosperm-specific expression cassette. The synthetic codon-optimized human serum albumin-epidermal growth factor gene (SEQ ID NO.1) was digested with Myyl and XhoI and cloned into pOsPMP02 to construct plasmid pOsPMP768, such as fig...

Embodiment 2

[0046] Example 2 Preparation of recombinant human serum albumin-EGF (OsrHSA-EGF) fusion protein crude extract

[0047] The OsrHSA-EGF transgenic rice seeds prepared in Example 1 were dehulled into semi-polished rice, and ground into 80-100 mesh rice flour. Mix the rice flour and the extract at a ratio of 1:5 (kg / L), stir and extract at 60°C for 1.5h, the components of the extraction buffer are 5-20mM phosphate, 5-20mM sodium acetate, 10-30mM ammonium sulfate, 10 ~20mM sodium octanoate, 1~5mM reduced glutathione, pH 7.0~7.5. Adjust the pH of the mixture of the above-mentioned rice flour and extract to 4.5 with acetic acid, stir and settle for 4 hours, add 2-5% perlite for pressure filtration or centrifugation, and then filter through a 0.22 μm membrane to obtain the crude extraction of OsrHSA-EGF liquid, see Image 6 with Figure 7 .

Embodiment 3

[0048] Embodiment 3 screens as thick and pure cationic chromatography filler

[0049] Comparing GE's SP Spharose Fast Flow, SP Spharose HP, Capto MMC, Borgron's SP FF, SP HP, Diamond MMC, and Nano-micro's Nano Gel 50sp, a total of 7 cationic fillers, it was found that the balance The buffer is 20mM NaAC, pH 5.0, when loading 5 times the column volume, when GE’s filler SPFF, SP HP and Navitas’s filler Nano Gel 50sp are used as the chromatographic medium, the target protein has a slight flow-through ; The target protein can be completely hung on the column of Borgron's filler SP FF and SP HF. However, in the case of linear elution with NaCl, none of the above single-mode chromatographic media can achieve ideal purification effects, especially the 50kD impurity band of rice seed host protein. In the further single-mode purification medium chromatography It is difficult to completely separate the target protein from the impurity protein, and the loading amount is limited within 5...

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Abstract

The invention discloses a chromatographic method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds. The methodcomprises the following specific steps: 1) extracting a crude extract containing recombinant human serum albumin-epidermal growth factor fusion from recombinant human serum albumin-epidermal growth factor fusion protein genetically engineered rice seeds; 2) performing cation exchange chromatography on the crude extract containing the human serum albumin-epidermal growth factor fusion protein to obtain a primary product I; and 3) carrying out anion exchange chromatography on the primary product I to obtain the purified human serum albumin-epidermal growth factor fusion protein target substance.According to the extraction and purification method disclosed by the invention, the recombinant human serum albumin-epidermal growth factor fusion protein of which the purity is greater than 95% canbe obtained, the protein activity is good, and the yield is high. The method is simple in process, economical, efficient and suitable for industrial production and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds. Background technique [0002] Human epidermal growth factor (human epidermal growth factor, hEGF) is a single-chain polypeptide containing 53 amino acids. By binding to receptors on receptor cells, it can widely stimulate a variety of cells such as epidermal cells, fibroblasts, Proliferation, differentiation and migration of endothelial cells and smooth muscle cells, thereby accelerating tissue regeneration and repair. In recent years, hEGF has been widely used clinically, such as promoting the healing of skin wounds, cuts, burns, scalds, oral ulcers, gastric ulcers and duodenum. In the high-end cosmetics market, there is also a huge demand. [0003] At present, hEGF is mainly prepared by human extraction, chemical synthesi...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/82C07K1/18C07K1/16
CPCC07K14/765C07K14/485C12N15/8257C07K2319/00Y02A50/30
Inventor 杨代常欧吉权邓贝
Owner WUHAN HEALTHGEN BIOTECHNOLOGY CORP
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