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Recombinant insulin secretion promoter fusion protein and its preparation method and use

A technology for promoting insulin and fusion proteins, which is applied in the field of preparation of recombinant fusion proteins, can solve the problems of no obvious correlation of antiviral responses and side effects, and achieve the effects of long half-life, good efficacy and good application prospects

Active Publication Date: 2016-01-20
CHENGDU JINXINHENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is 1.7% that the patient produces antibody to HSA after medication, but there is HSA antibody to reach 3.4% in the unmedicated person, and there is no obvious correlation (OsbornBL etc., JPharmacolExpTher, 2002,303(2):540-548;HumanGenomeSciencesReportsPositiveResultsofPhase1 / 2ClinicalTrialofAlbuferoninChronicHepatitisC.HGSIPress,2004,Nov.2;HumanGenomeSciencesReportsPositiveResultsofPhase2ClinicalTrialofAlbuferoninTreatmentNaivePatientswithChronicHepatitisC.HGSIPress,2005,Apr.14)

Method used

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  • Recombinant insulin secretion promoter fusion protein and its preparation method and use
  • Recombinant insulin secretion promoter fusion protein and its preparation method and use
  • Recombinant insulin secretion promoter fusion protein and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1, Construction of Engineering Strain PP-HE Expressing Recombinant Insulin Secretin Fusion Protein

[0069] 1. The source and design of the sequence

[0070] The insulin secretagogue is connected to the C-terminus of human serum albumin through a connecting peptide, and the structural formula of the fusion protein is as follows: human serum albumin-connecting peptide-insulin secretagogue (abbreviated as H-L-E, wherein, H represents coding human serum albumin nucleotide sequence or amino acid sequence, L represents the nucleotide sequence or amino acid sequence encoding the connecting peptide, E represents the nucleotide sequence or amino acid sequence encoding insulin secretagogue), and the recombinant insulin secretagogue fusion protein is called human Serum albumin-insulin secretagogue fusion protein (abbreviated as HSA-Ex4, or HE), the fusion protein produced by DNA recombinant technology is called recombinant human serum albumin-insulin secretagogue fusion p...

Embodiment 2

[0081] Example 2 Construction of Engineering Strain PP-PP-EHE Expressing Recombinant Insulin Secretin Fusion Protein

[0082] Insulin secretagogue-human serum albumin-insulin secretagogue fusion protein (EHE) is connected with an Exendin-4 at the N-terminus and C-terminus of HSA respectively through a connecting peptide, its amino acid sequence is shown in SEQ ID NO: 9, and its DNA The sequence is shown in SEQ ID NO:10.

[0083] As mentioned above, in order to facilitate purification, a His-tag tag and a natural enterokinase cleavage site are added before the target protein EHE, and the expressed protein structure obtained is: Histag-EK-E-L-HSA-L-E, as shown in SEQ ID NO: 11 Show. According to the codon preference of yeast, the DNA sequence encoding Histag-EK-E-L-HSA-L-E was designed, as shown in SEQ ID NO:12.

[0084] Using the method described in Example 1, the EHE expression strain PP-EHE was constructed.

Embodiment 3

[0085] Embodiment 3, the fermentation of engineering bacterium

[0086] This embodiment takes the expression strain PP-EHE as an example for specific illustration.

[0087] Primary seed culture: Inoculate the strain PP-EHE in 50mLYPD medium (Chinese name is yeast extract powder peptone glucose medium), culture at 250rpm, 30°C for about 20 hours.

[0088] Secondary seed culture: Inoculate the primary seed fungus culture solution in 200mL BMGY medium (a medium commonly used in the art, prepared according to conventional methods) at an inoculum size of 1% by volume fraction, and cultivate at 250rpm at 30°C for about 20 hours.

[0089] Fermentation culture: add 3.5LFM22 medium (containing 42.9g / LKH 2 PO 4 , 5g / L (NH 4 ) 2 SO 4 , 1.0g / LCaSO 4 2H 2 O, 14.3g / LK 2 SO 4 , 11.7g / LMgSO 4 ·7H 2 (0, 40g / L glycerol, the pH of the medium is 5.3), after autoclaving, add 200mL secondary seed bacteria and 3.5mL trace element solution (containing 2.0g / LCuSO 4 ·5H 2 O, 0.08g / LNaI, 3....

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Abstract

The invention relates to a recombinant insulin secretion promoter fusion protein. A polypeptide zone of the recombinant insulin secretion promoter fusion protein comprises a human serum albumin and an insulin secretion promoter. The insulin secretion promoter in a form of one or more monomers is connected to the human serum albumin in series by a connecting peptide. An amino terminal and / or a carboxyl terminal of the insulin secretion promoter are connected to a carboxyl terminal and / or an amino terminal of the human serum albumin by a connecting peptide. The number of the amino acid sequences for coding the connecting peptides is equal to the number of the amino acid sequences for coding the insulin secretion promoter. The recombinant insulin secretion promoter fusion protein has the advantages of good drug effects, long half life, simpleness, high efficiency, safety and good application prospect.

Description

technical field [0001] The present invention relates to a preparation process of a recombinant fusion protein, in particular to a recombinant insulin secretagogue fusion protein, a preparation method and its application. The recombinant insulin secretagogue fusion protein is the fusion of insulin secretagogue and human serum albumin Protein, the recombinant insulin secretagogue fusion protein as a long-acting drug for diabetes and obesity. Background technique [0002] Insulin secretagogue (English name is Exendin-4) is a kind of polypeptide (Eng, J. etc., J.Biol.Chem. , 265:20259-62, 1990; Eng, J. et al., J. Biol. Chem., 267:7402-05, 1992). Its amino acid sequence has 53% homology with glucagon-like peptide (GLP-1) (Coke et al., J. Biol. Chem., 268:19650-55, 1993). Chen and Drucker cloned the gene and concluded that it is the expression product of a gene other than the glucagon gene (GLP-1 is processed from proglucagon) (J. Biol. Chem., 272:4108-15, 1997). [0003] GLP,...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/38A61K38/22A61K47/48A61P3/10A61P3/04
Inventor 张仁怀孙朝国潘仲李鹏飞
Owner CHENGDU JINXINHENG BIOTECH CO LTD
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