Recombinant expression method and application of Brevinin-2GUb polypeptide

An expression method and expression vector technology, applied in the field of recombinant expression of Brevinin-2GUb polypeptide, can solve the problems of high artificial synthesis cost, difficult extraction, complex procedures, etc., achieve significant insulin secretion-stimulating activity, low cost, and simple expression and purification Effect

Inactive Publication Date: 2019-09-06
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the complicated procedures for isolating and purifying Brevinin-2GUb from frog skin secretions, the yield is very low, and the extraction is difficult; artificially synthesizing biologically active peptides is another important means to quickly obtain peptides, but the cost of artificial synthesis is high. Each amino acid is about 200 yuan

Method used

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  • Recombinant expression method and application of Brevinin-2GUb polypeptide
  • Recombinant expression method and application of Brevinin-2GUb polypeptide
  • Recombinant expression method and application of Brevinin-2GUb polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction of recombinant expression vector pET32a-Trx-His-EK-Brevinin-2GUb

[0040] (1) According to the gene sequence of Brevinin-2GUb, synthesize its DNA sequence in Gene Synthesis Company (Shanghai Shenggong), design the following primers (Bre-F / Bre-R) to amplify the target gene of Brevinin-2GUb:

[0041]

[0042] (2) Using the synthetic plasmid (pUC57-Brevinin-2GUb) containing the Brevinin-2GUb gene as a template, use the above amplification primers to amplify the Brevinin-2GUb target gene. The specific steps are as follows:

[0043] a. PCR reaction system (50μL):

[0044]

[0045] b. PCR amplification reaction program:

[0046]

[0047] (3) After the PCR reaction is completed, perform 2% (w / v) agarose gel electrophoresis identification to obtain a gene fragment containing Brevinin-2GUb with a size of about 133bp. Purify and recover the PCR product to obtain Brevinin-2GUb Gene fragments, then prepare a linear amplification reaction system, insert the recovered ...

Embodiment 2

[0061] Example 2: Expression and optimization of Trx-His-EK-Brevinin-2GUb fusion protein

[0062] (1) Transform the recombinant expression vector pET32a-Trx-His-EK-Brevinin-2GUb constructed in Example 1 into E. coli BL21 (DE3) competent cells by chemical transformation, and pick a single clone on the transformed plate for inoculation Into 6 mL of ampicillin-resistant LB liquid medium, cultured overnight at 37°C and 220 rpm as a seed solution. The seed solution was inoculated into 6 test tubes containing 6 mL of fresh ampicillin-resistant LB liquid medium at a ratio of 1:100 (v / v), and incubated at 37°C and 220 rpm. 600 =0.6~0.8, 3 tubes were induced by adding 1mM IPTG, and the other 3 tubes were not added with inducer IPTG as control. One tube was induced and 1 tube was not induced. A total of three groups were induced at 16℃, 25℃, 37℃ for 20h. , 12h, 5h for expression temperature optimization.

[0063] (2) After the induction of expression, the OD of the expressed bacterial solut...

Embodiment 3

[0066] Example 3: Mass expression of Trx-His-EK-Brevinin-2GUb fusion protein and purification by nickel ion affinity chromatography

[0067] (1) The single clone in Example 2 was inoculated into 8 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm as a seed solution. The seed solution was inoculated into 600 mL of fresh ampicillin-resistant LB liquid medium at a ratio of 1:100 (v / v), and incubated at 37°C and 220 rpm. 600 =0.6~0.8, add the inducer IPTG to a final concentration of 1mM, and then place it in a shaker at 16℃ to induce expression for 20h.

[0068] (2) After the expression, the cells were collected by centrifugation at 6000 rpm for 10 minutes, resuspended in purified Buffer A (20mM Tris, 500mM NaCl, 20% glycerol, 20mM imidazole, pH 8.5), crushed by high pressure, centrifuged to collect the supernatant, and then proceeded Purify by nickel ion affinity chromatography, collect the passing fluid sample and the samples before and after purification for SDS-PAGE...

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Abstract

The invention belongs to the field of genetic engineering and biopharmaceuticals, and discloses a recombinant expression method of a Brevinin-2GUb polypeptide, wherein the method comprises the steps:(1) inserting a Brevinin-2GUb gene into pET32-series vectors containing thioredoxin, histone labels and enterokinase digestion sites, to obtain recombinant expression vectors; (2) transforming the recombinant expression vectors into escherichia coli to obtain recombinant expression engineering strains; (3) carrying out inducible expression on the engineering strains, centrifuging to collect bacteria, crushing, centrifuging to obtain a supernatant, and carrying out nickel ion affinity chromatography on the supernatant to obtain a fusion protein; and (4) dialysing the fusion protein with an enterokinase digestion buffer, then cutting the fusion protein by enterokinase, carrying out nickel ion affinity chromatography, then concentrating to obtain the target polypeptide without labels. The polypeptide can significantly promote insulin secretion activity in INS-1 cells at low concentration, and can be used for preparing hypoglycemic polypeptide drugs.

Description

Technical field [0001] The invention belongs to the technical fields of genetic engineering and biopharmaceuticals, and specifically relates to a method and application for recombinant expression of Brevinin-2GUb polypeptide. Background technique [0002] In recent decades, the global prevalence of diabetes in adults has continued to rise (Ogurtsova K.IDF DiabetesAtlas:Global estimates for the prevalence of diabetes for 2015and 2040[J].Diabetes Research and Clinical Practice,2017,128:40-50) . With economic development and the improvement of people's living standards, diabetes has become one of the most common chronic diseases worldwide. According to the International Diabetes Federation (IDF), there were 451 million (18-99 years old) diabetes patients worldwide in 2017, and it is estimated that the number of diabetes patients will increase to 693 million by 2045 (Cho NH.IDF Diabetes Atlas: Global estimates of diabetesprevalence) for 2017 and projections for 2045[J].Diabetes Res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C07K14/46C07K1/22A61K38/17A61K9/08A61P3/10
CPCC12N15/70C12N15/66C07K14/463A61K9/0095A61P3/10A61K38/00
Inventor 王菊芳林静莲马毅
Owner SOUTH CHINA UNIV OF TECH
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