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Dual blood sugar and blood fat adjusting fusion protein and preparation method and application thereof

A fusion protein and protein technology, applied in the field of host cells, can solve the problems of short plasma half-life and direct application, etc.

Active Publication Date: 2011-03-30
CHONGQING PEG BIO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although natural GLP-1 has many advantages in the treatment of diabetes, its short plasma half-life in vivo limits its direct clinical application

Method used

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  • Dual blood sugar and blood fat adjusting fusion protein and preparation method and application thereof
  • Dual blood sugar and blood fat adjusting fusion protein and preparation method and application thereof
  • Dual blood sugar and blood fat adjusting fusion protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1E

[0047] Example 1 Exendin4-FGF21 fusion protein recombinant expression and preparation in Escherichia coli

[0048] In this example, the involved fusion protein of GLP-1 and FGF-21 has the structure of Met-Exendin4-GGGGGS-FGF21. According to the amino acid sequences of Exendin-4 and FGF-21, and according to the codons preferred by Escherichia coli, Bao Biology (Dalian) Co., Ltd. was entrusted to carry out the whole gene synthesis. The nucleotide sequences are:

[0049] cDNA sequence of Fgf-21 (SEQ ID NO: 4)

[0050] CAC CCA ATC CCA GAT TCT AGT CCA CTG TTA CAA TTC GGA GGT CAG

[0051] GTT CGT CAA CGT TAT CTG TAT ACC GAT GAC GCA CAG CAG ACC GAA GCC CAT

[0052] TTG GAA ATT CGT GAA GAT GGA ACC GTT GGT GGT GCT GCC GAT CAA TCT CCT

[0053] GAA TCA CTG TTA CAG CTG AAA GCA TTG AAA CCA GGA GTT ATT CAG ATT CTG

[0054] GGT GTC AAA ACC TCT CGT TTT TTA TGT CAA CGT CCT GAC GGT GCC CTG TAT

[0055] GGA AGT TTG CAT TTT GAT CCA GAA GCC TGT TCT TTT CGT GAA CTG TTA CTG

[0056] GAA GAT GG...

example 2

[0079] Example 2 expresses and purifies Exendin4-FGF21 protein in yeast

[0080] The correct recombinant plasmid pET-3C-Exendin4-FGF21 identified in Example 1 is used as a template, and its PCR fragment containing Xho I and EcoR I restriction sites is amplified with primers. pIC9k was ligated to obtain the recombinant plasmid pPIC9K-Exendin4-FGF21. The recombinant plasmid was linearized with Sac I, and the linearized recombinant plasmid pPIC9K-Exendin4-FGF21 was introduced into GS115 Pichia pastoris (Invitrogen) with an electroporator, and was screened by His+ and Zeocin to obtain a high-copy clone resistant to Zeocin 1mg / ml bacteria. Pick expression bacteria, inoculate in 5ml YPD medium, culture overnight at 30°C, transfer to 250ml BMGY medium. Cultivate until the OD600 is 2-3, collect the bacteria, dilute with carbon-source-free BMMY medium to resuspend (OD600 is 5-6), and continue to cultivate. Methanol was added once every 24 hours, and methanol was added to a final con...

example 3

[0081] Example 3 Detection of fusion protein Exendin4-FGF21 binding to GLP-1 receptor

[0082] Oral glucose tolerance test: take 8-10 weeks of Balb / c mice (Chongqing Medical University Animal Center), and randomly divide them into three groups: blank control group, fusion protein injection group and Exendin-4 (provided by Chengdu Xunti Biotechnology Co., Ltd. Synthetic, hereinafter the same) control group. After 16-18 hours of fasting (without water) one day before the experiment, 25ug / kg fusion protein and 5ug / kg Exendin-4 (both with the same molar concentration of 1nM) were injected intraperitoneally. One hour after administration, 1.5 mg / g of body weight glucose was perfused, and blood was collected 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours after the administration of glucose to detect blood sugar levels. The results showed that the fusion protein had better hypoglycemic effect. Compared with the Exendin-4 control group, it still has an obvious hypoglycemic effect...

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Abstract

The invention relates to a dual blood sugar and blood fat adjusting fusion protein and a preparation method and application thereof. The invention provides the fusion protein of a human fibroblast growth factor 21 (FGF-21), a glucagon similar peptide 1 (GLP-1) and an analog thereof (Exendin-4), a DNA sequence for coding the fusion protein, a vector containing the DNA sequence, a host cell containing the vector, a method for preparing the fusion protein by using gene engineering, and a medicinal composition of the fusion protein. The fusion protein of the invention has the activities of the FGF-21 and the GLP-1 at the same time, and can be used as a treatment medicament of obesity, II-type diabetes and hyperlipidemia.

Description

technical field [0001] The invention relates to the fields of DNA recombination technology and medicine. More specifically, the present invention relates to a fusion protein of human fibroblast growth factor 21 (FGF-21) and human glucagon-like peptide 1 (GLP-1) and its analog (Exendin-4), encoding the fusion protein The DNA sequence, the vector containing the DNA sequence, and the host cell containing the vector. The method for preparing the fusion protein by genetic engineering, and the application of the fusion protein in the treatment of diseases such as obesity, type 2 diabetes and hyperlipidemia. Background technique [0002] FGF-21 [0003] Fibroblast growth factors are a class of multifunctional active polypeptides, and 18 mammalian fibroblast growth factors (FGF1-FGF10, FGF16-FGF23) have been discovered so far. According to sequence homology, they can be subdivided into 6 different subfamilies (Andrew Beenken et al., Nat Rev Drug Discov, 2009, 8(3):235-253). Fibr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12N1/19C12N5/10C12P21/02A61K38/26A61P3/04A61P3/10A61P3/06C12R1/19
CPCC07K2319/00C07K14/50C07K14/605A61K38/00A61P3/04A61P3/06A61P3/10
Inventor 范开赵志全张淳陈勇罗华杨黎刘洪洪
Owner CHONGQING PEG BIO BIOTECH CO LTD
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