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Recombinant expression method for hypoglycemic polypeptide Aglycin and application thereof

An expression method and hypoglycemic technology, which is applied in the fields of genetic engineering and biopharmaceuticals, can solve the problems of cumbersome process and low efficiency of extraction and separation, and achieve the effect of easy aggregation, efficient acquisition, and small molecular weight

Active Publication Date: 2019-08-02
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, Aglycin is mainly prepared by extraction and separation of soybean or pea seeds. The extraction and separation efficiency is low and the process is very cumbersome. It needs to go through acetic acid extraction, alginic acid adsorption, dextran G-25 gel filtration, ion exchange chromatography, Multiple steps such as reversed-phase high-performance liquid chromatography preparation

Method used

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  • Recombinant expression method for hypoglycemic polypeptide Aglycin and application thereof
  • Recombinant expression method for hypoglycemic polypeptide Aglycin and application thereof
  • Recombinant expression method for hypoglycemic polypeptide Aglycin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of recombinant expression vector pET30a-Aglycin-Mxe-PT-ELK16

[0039] (1) According to the gene sequence of Aglycin, its DNA sequence Ag was synthesized at Gene Synthesis Company (Shanghai Sangong), and the following primers (Ag-F / Ag-R) were designed to amplify the Aglycin target gene:

[0040]

[0041] (2) Using the synthesized plasmid (pUC57-Ag) containing the Aglycin gene as a template, use the above amplification primers to amplify the Aglycin target gene. The specific steps are as follows:

[0042] a. PCR reaction system (50μL):

[0043]

[0044] b.PCR amplification reaction procedure:

[0045]

[0046] (3) After the PCR reaction finishes, carry out 1% (w / v) agarose gel electrophoresis identification, obtain the gene fragment that contains Aglycin about 165bp in size, purify and reclaim the PCR product, obtain the gene fragment that contains Aglycin, then Prepare a linear amplification reaction system, and insert the recovered Agl...

Embodiment 2

[0059] Example 2: Expression and optimization of Aglycin-Mxe-PT-ELK16 recombinant protein

[0060] (1) Transfer the recombinant expression vector pET30a-Aglycin-Mxe-PT-ELK16 constructed in Example 1 into Escherichia coli BL21(DE3) competent cells by chemical transformation, pick a single clone on the transformation plate and inoculate it into 6mL The LB liquid medium was cultured overnight at 37°C and 220 rpm as the seed solution.

[0061] a. Temperature optimization of Aglycin-Mxe-PT-ELK16 expression: Inoculate the seed liquid into 6 test tubes containing 6 mL of fresh LB liquid medium at a ratio of 1:100 (v / v), place at 37°C, 220rpm cultured, when OD 600 When =0.6~0.8, 3 branches were added with 1mM IPTG for induction, and the other 3 branches were not added with inducer IPTG as a control, paired in pairs (1 branch was induced, 1 branch was not induced) to induce expression at 16°C, 25°C, and 37°C, respectively 24h, 12h, 5h.

[0062] b. Optimizing the concentration of the...

Embodiment 3

[0068] Embodiment 3: DTT cleavage condition optimization of Aglycin-Mxe-PT-ELK16 protein

[0069] (1) Inoculate the single clone in Example 2 into 8 mL of LB liquid medium for overnight culture at 37° C. and 220 rpm as the seed solution. Inoculate the seed liquid into 600mL fresh LB liquid medium at a ratio of 1:100 (v / v), place it at 37°C, and cultivate it at 220rpm, when the OD 600 =0.6~0.8, add the inducer IPTG to a final concentration of 0.2mM, and then place in a shaker at 16°C to induce expression for 24h.

[0070] (2) After the expression, the cells were collected by centrifugation at 9000rpm for 10 min, resuspended in LysisBuffer, crushed by high pressure, and the precipitate was collected by centrifugation, and washed by adding washing buffer to the precipitate to obtain a precipitate sample.

[0071] a. Add the corresponding volume (20OD / mL) of cutting solution containing 40mM DTT to the precipitated samples, suspend evenly, place at 4°C for cutting, then take sampl...

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Abstract

The invention belongs to the technical field of genetic engineering and bio-pharmaceuticals and specifically discloses a recombinant expression method for a hypoglycemic polypeptide Aglycin and an application thereof. The recombinant expression method comprises the following steps: constructing a recombinant expression vector capable of in vitro self-shearing, by connecting Aglycin with a self-assembling peptide by a self-shearing intein and a connecting peptide; in vivo expressing the recombinant expression vector in escherichia coli, thereby acquiring a recombinant expression strain; addingan inducer into the recombinant expression strain for inducing expression; centrifugally collecting thalli; breaking and centrifuging the thalli and then precipitating; cutting with the inducer and centrifuging, thereby acquiring a supernatant; purifying the supernatant after cutting, thereby acquiring high-purity Aglycin. The invention firstly adopts a genetic engineering method for realizing soluble expression of Aglycin. The polypeptide has a certain hypoglycemic activity for inhibiting alpha-glucosaccharase and can be used for preparing a hypoglycemic polypeptide drug.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and biopharmaceuticals, and in particular relates to a recombinant expression method and application of the hypoglycemic peptide Aglycin based on self-assembling peptides and self-cleaving peptides. Background technique [0002] So far, diabetes (diabetes mellitus, DM) has become one of the hidden killers that threaten human life and health. According to a report released by the International Diabetes Federation (IDF), more than 425 million people worldwide suffered from diabetes in 2017. Among them, Chinese patients The number accounts for more than 25% of the world's total, up to 114 million (Mu Yoshiaki, Jia Weiping. Brief introduction to the research progress of diabetes in China [J]. Chinese Science: Life Science, 2018, 48(08): 807-809). Diabetes is a metabolic disorder syndrome caused by a variety of factors, among which type Ⅱ diabetes accounts for more than 90% of the total nu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C07K14/415A61K38/16A61P3/10
CPCC12N15/70C12N15/62C07K14/415A61K38/168A61P3/10C07K2319/735C07K2319/00Y02A50/30
Inventor 王菊芳黄敏华马毅林静莲
Owner SOUTH CHINA UNIV OF TECH
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