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A kind of recombinant active peptide and its synchronous preparation method

A technology of active peptides and peptide segments, applied in the field of genetic engineering, can solve the problems of lack of high in vivo activity and low cost, and achieve the effects of improving vascular endothelial cell function, increasing production rate, and broad application prospects

Active Publication Date: 2018-01-16
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have confirmed that hypotensive peptides and antioxidant peptides have different targets, different intervention systems and different mechanisms of action in the body, but they have positive effects on the prevention and control of hypertension and related cardiovascular diseases
However, there is no report on "using genetically engineered bacteria to simultaneously prepare blood pressure-lowering peptides and antioxidant peptides"
[0006] At present, there is a lack of a high in vivo activity, low-cost recombinant active peptide and its simultaneous preparation method

Method used

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  • A kind of recombinant active peptide and its synchronous preparation method
  • A kind of recombinant active peptide and its synchronous preparation method
  • A kind of recombinant active peptide and its synchronous preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Such as Figure 1 to Figure 7 as shown, figure 1 Schematic diagram of the recombinant plasmid constructed for the present invention; figure 2 It is a schematic diagram of the PCR identification of the recombinant plasmid of the present invention. Lane M is the DL10000 DNA standard sample, and lanes 1, 2, and 3 are all the same PCR product (using the recombinant plasmid pET28a-ESA3 as a template, primers 5'-TTCCTGGAACCGGA-3' and 5'- CCAAGCCACACGA AAC-3' is the upstream and downstream primers); image 3 It is a schematic diagram of the double enzyme digestion identification of the recombinant plasmid of the present invention, M is DNA Marker, and lanes 1-5 are Nco I / Bam HI, Bam HI / Hind III, Hind III / Xho I, Bam HI / Xho I, Nco I respectively / Xho I double digestion product, lane 6 is the recombinant plasmid; Figure 4 SDS-PAGE is a schematic diagram of the identification of recombinant protein inclusion body expression. Lane M is the protein standard sample, and lanes 1,...

Embodiment 2

[0079] The difference between Example 2 and Example 1 is that the engineering bacteria pET28a-ESA3 / E.coli BL21(DE3) was constructed according to the steps (1) and (2) in Example 1. Put a single colony of engineering bacteria in 20ml of LB medium containing 50μg / ml Kana, culture overnight at 37°C, 200rpm shaker, and then transfer the overnight culture solution into TB containing 50μg / ml Kana at an inoculum size of 2%. In the culture medium, 37°C, 200rpm, shake the flask to cultivate until the OD value of the bacteria reaches 0.6-0.8, add IPTG to the final concentration of 0.02mM, induce culture at 20°C for 20h, and sample for SDS-PAGE analysis.

[0080] Such as Figure 4 It is known that after the engineered bacteria were induced and cultured for 20 hours under the condition of low temperature and low inducer concentration, a protein band with a molecular weight of about 45 kDa appeared relative to the pre-induction sample; after the cells were ultrasonically disrupted, the sol...

Embodiment 3

[0082] The difference between Example 3 and Example 1 lies in the preparation of the recombinant small peptide mixture and its activity identification. Take an appropriate amount of prepared ELPs-SUMO-A3, add SUMO protease according to the addition amount of 3%, and enzymatically hydrolyze at 30°C for 5h. After the enzymolysis, add sodium chloride with a final concentration of 1.5mol / l to the enzymolysis solution, bathe in water at 30°C for 10 minutes, centrifuge at 8000r / min for 10 minutes, and keep the supernatant, which is the recombinant active peptide. Simulate the natural physiological digestion process of the human body in vitro, adjust the pH of the 2mg / ml recombinant active peptide solution to 2.0 with hydrochloric acid, add 2% pepsin, hydrolyze it at 37°C for 4 hours, stop the reaction in a boiling water bath, adjust the pH to 7.0, and take part of the hydrolyzed solution Determination of peptide activity. Add 2% trypsin (or a mixture of trypsin and chymotrypsin) to...

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Abstract

The invention discloses a recombinant active peptide and a synchronous preparation method thereof. The recombinant active peptide is formed by stacking or directional series of active peptide monomers, and the active peptide monomers include blood pressure-lowering peptide monomers and antioxidant peptides Monomer, its amino acid sequence is shown in the sequence listing SEQ ID NO: 1. A nucleotide sequence encoding the recombinant active peptide of the present invention is shown in the sequence listing SEQ ID NO: 2. The invention has low cost, the active peptide in the obtained recombinant polypeptide can be effectively released by pepsin, trypsin and chymotrypsin, and the prepared active peptide mixture has high antihypertensive activity and antioxidant activity in vivo.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant active peptide and its simultaneous preparation method. Background technique [0002] Bioactive peptides are generally stored in the parent protein sequence and do not have biological activity, but they can perform physiological functions in vivo after being released by proteases or food processing in vivo or in vitro. At present, functionally active peptides are mainly prepared by enzymatically hydrolyzing protein raw materials. At present, there have been many patent reports on the preparation of hypotensive peptides and antioxidant peptides by enzymatic hydrolysis. Compared with biological extraction, chemical degradation and chemical synthesis, enzymatic hydrolysis has the advantages of high product safety, mild production conditions, and little damage to protein nutritional value. However, the relevant active peptide products currently appearing in the dome...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/11C12N15/70A61K38/16A61P9/12A61P39/06
CPCA61K38/00C07K14/00
Inventor 饶胜其方维明杨振泉黄阿根高璐
Owner YANGZHOU UNIV
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