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Recombinant human serum albumin/pancreatic glucagon peptide fusion protein having blood sugar content continuous control function

A serum albumin and fusion protein technology, applied in the field of long-acting medicinal functions, can solve the problems of limiting the clinical application of GLP-1

Active Publication Date: 2016-08-24
BEIJING MEIFUYUAN BIO PHARM TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, the clinical application of GLP-1 also faces a problem, that is, the GLP-1 produced by the human body is easily degraded by dipeptidyl peptidase IV (DPP-IV) in the body, and its plasma half-life is less than 2 minutes. Continuous intravenous infusion or continuous subcutaneous injection is necessary to produce curative effect, which greatly limits the clinical application of GLP-1

Method used

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  • Recombinant human serum albumin/pancreatic glucagon peptide fusion protein having blood sugar content continuous control function
  • Recombinant human serum albumin/pancreatic glucagon peptide fusion protein having blood sugar content continuous control function
  • Recombinant human serum albumin/pancreatic glucagon peptide fusion protein having blood sugar content continuous control function

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Brief Description of Molecular Cloning Technology

[0077] Conventional molecular cloning techniques include extraction of DNA and RNA, agarose gel and polyacrylamide gel electrophoresis, connection of DNA fragments, and restriction endonuclease digestion reactions are all referred to literature (Maniatis, et al., "Molecular cloning experiment Handbook" published by Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982). The purification of plasmid DNA and the recovery of DNA fragments are all prepared by commercial purification columns. DNA polymerase chain reaction (PCR) (referring to literature Saiki et al., Science, 230: 1350, 1985) used enzymes and PCR instruments required for the reaction are all Perkin Elmer products. And refer to the manufacturer's operating procedures. The oligonucleotide primers required for DNA sequencing and DNA amplification are completed by specialized institutions. The transgenic Escherichia coli was purchased fr...

Embodiment 2

[0078] Embodiment 2: Human serum albumin (HSA) gene expression and the construction of carrier plasmid

[0079] The HSA cloning method and test results described by the inventor in its invention patents ZL02142881.6, ZL200410042814.8 and ZL200410057313.7 were used. The HSA sequence of GenBank retrieval number AY728024 is used for gene recombination fusion protein of HSA gene sequence and cell growth factor gene sequence in the present invention. Using AY728024 sequence to encode HSA can make the fusion protein obtain unexpected high-level expression in yeast. Expression plasmids (see Chinese patent ZL02142881.6) can also be used in the embodiments of the present invention. The recombinant human serum albumin expression vector plasmid DNA structure of pYZ-HSA is also in the Chinese invention patent ZL02142881.6 figure 2 Disclosed in and used to construct various molecular structure expression vector basic plasmids of the recombinant human serum albumin / glucagon peptide fusio...

Embodiment 3

[0080] Example 3: Synthesis of Human GLP-1 Tandem Double Gene Fragment and Recombinant Human Serum Albumin / Glucagon Peptide 2 Construction of Fusion Protein Expression Vector Plasmid

[0081] The inventor first synthesized the GLP-1 gene sequence using the GLP-1 wild-type amino acid sequence 7-36 peptide as a standard, and fused it with the human serum albumin gene to obtain various GLP-1 fusion proteins and carried out biological activity verification. Then, using the GLP-1 analogue amino acid sequence in Albugon of HGSI in the United States as a standard, the DNA sequence total synthesis method was used to artificially synthesize the double repeat sequence of human GLP-1 (7-36, Ala8Gly) as Seq No.3, corresponding to The amino acid sequence of is Seq No.4. The synthesized GLP-1 2 The N-terminal is equipped with a Bsu36I endonuclease site to achieve seamless connection and direct connection with the C-terminal of human serum albumin:

[0082] Seq No.3:

[0083] 5'-G CTTA...

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Abstract

The invention relates to a novel molecular drug structure of recombinant human serum albumin / pancreatic glucagon peptide fusion protein capable of obviously reducing blood sugar content in blood and having continuous effect: rGLP-12 / HSA / GLP-12. Especially, the invention uses a genetic engineering technology for constructing the engineered yeast strain to express the produced recombinant fusion protein. The recombinant fusion protein has the advantages that 1) blood sugar content in the blood is reduced, body weight is reduced, and the recombinant fusion protein is used for treating diabetes and cardiovascular disease; 2) life of pancreatic glucagon peptide is greatly prolonged in vitro and vivo, so that the recombinant fusion protein has maximum stability in the blood and can be continuously effected on the blood sugar control; and 3) saccharomycetes can be used for realizing low-cost large-scale production and preparation process.

Description

technical field [0001] The present invention is a continuation application of Chinese patents ZL02142881.6, ZL200410042814.8 and ZL200710057571.9. The invention relates to gene recombination and expressed human serum albumin fusion protein and a preparation method thereof, and relates to a single fusion protein or a method for combining different fusion proteins. In particular, it involves the expression and production of recombinant fusion proteins with different molecular structures formed by human serum albumin and glucagon peptides by yeast, which can be used to control the symptoms of hyperglycemia in patients with type 2 diabetes, preparations for weight loss and prevention of cardiovascular diseases And treatment and long-acting medicinal function with continuous effect. Background technique [0002] Human serum albumin (HSA) is a soluble monomeric protein that constitutes half of the total protein in blood. As a basic carrier, albumin carries molecules such as fatt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/85C12N15/81C12N15/82A61K38/38A61K38/26A61P3/10A61P3/04
Inventor 富岩于在林
Owner BEIJING MEIFUYUAN BIO PHARM TECH
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