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GLP-1 analogue-COL3A1 fusion protein

A GLP-1, fusion protein technology, applied in the field of medicine

Inactive Publication Date: 2019-08-06
SHANGHAI HUIMMUTECH BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Fc segment of IgG in dulaglutide may have ADCC (Antibody-Dependent Cell Cytotoxicity, antibody-dependent cell-mediated cytotoxicity), which has potential immunogenicity and side effects

Method used

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  • GLP-1 analogue-COL3A1 fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Example 1: Construction of DNA encoding GLP-1-COL3A1 fusion protein

[0135] Coding fusion protein GLP-1-2L-COL of the present invention 598-896 The gene (SEQ ID NO: 8) was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and cloned into the pUC57 plasmid. The 5' end of the fusion gene contains an XhoI restriction site, and the 3' end contains a TAA stop codon and NotI restriction enzyme site, the pUC57 plasmid was named pUC57-GLP-COL-1.

[0136] Use restriction endonucleases XhoI and NotI (purchased from Fermentas) to carry out double enzyme digestion on pUC57-GLP-COLA-1 according to the instruction manual, and produce the encoded GLP-1-2L-COL with a length of about 1050bp after digestion. 598-896 The gene fragment of the fusion protein was recovered by gel (the gel recovery kit was purchased from Axygen). At the same time, pPic9m was digested with XhoI and NotI, and the digested plasmid band with a length of 9000 bp was gel recovered.

[0137] The fusion pr...

Embodiment 2

[0139] Embodiment 2: Expression of heterologous fusion protein

[0140] The vector inserted with the fusion protein gene in Example 1 was extracted, and transformed into Pichia pastoris GS115 competent cells by electroporation. After screening recombinants by nutrient-limiting medium, G418 resistance was used to screen for high-copy recombinants. Finally, recombinant Pichia pastoris cells containing heterologous fusion protein genes suitable for recombinant expression are obtained.

[0141] Recombinant Pichia pastoris cells were inoculated into a 5L fermenter for small-scale preparation after seed amplification, and the fermentation lasted for 5 days, in which methanol was used to induce the expression of heterologous fusion proteins, and the induction lasted for 36 hours. collected for protein purification.

[0142] Table 1: Composition of Pichia pastoris GS115 seed expansion medium

[0143] formula content Yeast Extract 10.0g / L Peptone 20.0g / L ...

Embodiment 3

[0146] Embodiment 3: Purification of heterologous fusion protein

[0147] Two preferred heterologous fusion proteins GLP-1-2L-COL 598-896 , GLP-1-2L-COL 733-896 A similar purification procedure was employed.

[0148] The 4L fermentation medium was collected using a PALL 0.2μm hollow fiber filtration system to obtain about 4L supernatant. The supernatant was then subjected to sample ultrafiltration with a PALL 50kDa filter membrane ultrafiltration system to remove some impurity proteins. The ultrafiltered liquid was finally purified by Source30Q anion exchange chromatography, and the sample was eluted with a linear gradient of 0-500mM NaCl, and finally the purified heterologous fusion protein was obtained. SDS-PAGE was used to determine the purity and molecular weight of the heterologous fusion protein, the purity was >90%, and the molecular weight was in line with expectations ( image 3 ).

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Abstract

The invention relates to a GLP-1 analogue-COL3A1 fusion protein. Specifically, the fusion protein comprises the glucagon-like peptide 1 analog and a human type III collagen alpha1 chain fusion protein, and the fusion protein has the hypoglycemic effect of the glucagon-like peptide 1 and has an extended half-life in vivo.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a GLP-1 analogue-COL3A1 fusion protein. Background technique [0002] Diabetes mellitus is a serious chronic disease mainly caused by hyperglycemia and insufficient secretion and loss of function of endogenous insulin. Diabetes can cause multiple complications, such as the vascular system, kidneys, retina, lens, peripheral nerves, and skin, which can affect life expectancy and quality of life. With the development of social economy and the improvement of living standards in various countries in the world, the incidence and prevalence of diabetes are increasing year by year. Diabetes has become the third chronic disease that seriously endangers human health after tumors and cardiovascular and cerebrovascular diseases. Diabetes is basically divided into four categories, including: type Ⅰ (insulin-dependent), type Ⅱ (non-insulin-dependent), other types and gestational diabetes. Among them,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/26A61P3/10
CPCC07K14/605C07K14/78C07K2319/00A61K38/00
Inventor 陈国友
Owner SHANGHAI HUIMMUTECH BIOTECHNOLOGY CO LTD
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