Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Yeast for dietary therapy of diabetes and construction method thereof

A technology of diabetes and Saccharomyces cerevisiae, which is applied in the field of diabetic dietary yeast and its construction, and can solve the problems of inability to realize clinical application, short half-life of natural GLP-1, etc.

Inactive Publication Date: 2010-09-08
NANKAI UNIV
View PDF4 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the degradation of dipeptidyl peptidase (DPP-IV) in plasma, the half-life of natural GLP-1 in plasma is very short, so it cannot be used clinically.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Yeast for dietary therapy of diabetes and construction method thereof
  • Yeast for dietary therapy of diabetes and construction method thereof
  • Yeast for dietary therapy of diabetes and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0038] Example 2. Construction of the integration vector pNK-GLP.

[0039] Using the multi-copy tandem rDNA sequence in the chromosome of Saccharomyces cerevisiae as the site for multi-copy integration of exogenous genes, two pairs of primers were designed for a 2.2 kb homologous recombination hotspot region between 25S and 5S rDNA in the rDNA sequence:

[0040] rDNA Primer 1 (A): 5′-ACC GAGCTC GCATGCCACCTACCGACC-3′

[0041] rDNA Primer 2 (B): 5′-GAG GAATTC GTTAACAGGACATGCCTTTG-3′

[0042] rDNA Primer 3 (C): 5′- CAACTG CAGGTTAACTATAGGAAATGAG-3′

[0043] rDNA Primer 4(D): 5′-ACA CTGCAG GGGTTTAGACCGTCGTGAGAC-3′

[0044] The underlined parts are the restriction sites of Sac I, EcoR I, Pst I and Pst I respectively, using S. The PCR reaction was carried out in a mixture of 0.5 μl, 0.5 μl 10 mM dNTPs and 15 μl double distilled water. The conditions were: denaturation at 94°C for 10 min, denaturation at 94°C for 50 s, annealing at 55°C for 1 min, extension at 72°C for 90 ...

Embodiment 3

[0052] Example 3. Construction of recombinant Saccharomyces cerevisiae stably expressing rolGLP-1.

[0053] The vector pNK-GLP was linearized by HpaI digestion and transformed into Saccharomyces cerevisiae BJ2407. The transformant was screened in the auxotrophic medium not containing uracil, and the transformant SG2 in which rolGLP-1 and URA3 were integrated into the yeast chromosome was obtained.

[0054] SG2 genomic DNA was extracted and digested with BglII, and the amplified product of the rolGLP-1 monomer nucleotide sequence was used as a probe for Southern hybridization. The result was that there was no hybridization band in the genome lane of the recipient strain BJ2407, while in the selected recombinant yeast SG2 hybridization bands appeared. image 3 Among them, lane 1 is the genome digestion product of the recipient strain BJ2407; lane 2 is the digested product of the positive control pMD-GLP; lane 3 is the digested product of pNK1; lane 4 is the recombinant yeast SG2...

Embodiment 4

[0056] Embodiment 4, hypoglycemic biological activity test.

[0057] After fasting for 10 hours, Wistar rats (Experimental Animal Center, Chinese Academy of Military Medical Sciences) were intraperitoneally injected with 60 mg / kg streptozotocin to construct hyperglycemia model rats. Thirty hyperglycemic rats (25.1±3.5mM) were divided into 5 groups, and 5g of recombinant yeast SG2 / kg was administered to the stomach; 0.5g of recombinant yeast SG2 / kg was administered to the stomach; Stomach; the positive control group received 0.1g metformin / kg intragastric administration; the model group received 5g yeast BJ2407 / kg intragastric administration, and all treatments lasted for 20 days. Before measuring blood glucose, the rats were fasted for 10 h without water. Blood was collected from the tail vein, and the fasting blood glucose value was measured using a micro blood glucose tester (Advantage blood glucose meter), and the measurement results were as follows: Image 6 As shown, it...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention describes a yeast for the dietary therapy of diabetes and a construction method thereof. A saccharomyces cerevisiae genome DNA as a template is used for amplifying two sections of rDNA sequences; pYX212 as a template is used for amplifying a nutritional marker gene URA3 and an expression cassette segment driven by a triosephosphate isomerase promoter; the four amplified products are respectively cloned at corresponding loci of pUC18, and thereby an integrating vector pNK1 is obtained. A DNA synthesization point mutation technique is then utilized to remove the restriction sites of dipeptidase VI and trypsin in the natural GLP-1 molecule, so that type I primer segments, type II primer segments and left and right connected filling primer segments are synthesized, and a ten-repeat tandem rolGLP-1 sequence is obtained by the method of step-by-step annealing and one-step connection, and is cloned in the pNK1, so that pNK-GLP is obtained. The pNK-GLP is then linearized, saccharomyces cerevisiae BJ2407 is transformed, and after screening, recombinant yeast SG2 is obtained. Because the rolGLP-1 gene is integrated in yeast chromosome, the recombinant strain can stably and efficiently express the characteristics of the rolGLP-1 and reduce the blood sugar level of a rat with hyperglycaemia, and can be used in the development of yeast biomedicine for treating diabetes.

Description

technical field [0001] The invention relates to a dietary yeast for diabetes and a construction method thereof, belonging to the field of biotechnology. Background technique [0002] Diabetes is a metabolic disorder that seriously threatens human health and life. As far as the number of people with diabetes is concerned, it is already close to the epidemic proportion. At present, in addition to chemical drugs such as sulfonylureas, metformin, acarbose, and thiazolidinediones, the drugs used to treat type 2 diabetes also include peptide drug insulin. They lower blood sugar through different mechanisms, but these drugs do not prevent beta cell death or promote beta cell regeneration. Therefore, it is still unavoidable for patients with type 2 diabetes to continue to decline in β-cell function if they are treated with the above methods. [0003] Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone processed and secreted from intestinal L cells, and it is expected to beco...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N1/19C12N15/81A61K36/064A61P3/10C12R1/865
Inventor 李明刚武志强赵磊廖芳李娜马百成胡晓宇王维婧卫一鸣陈苗王瑞菊
Owner NANKAI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com