Recombinant GLP-1 analogue Fc fusion protein optimized gene and application thereof

A GLP-1, fusion protein technology, applied in the direction of animal/human protein, fusion polypeptide, recombinant DNA technology, etc., can solve problems such as long-term maintenance of efficacy, lack of stability of cell lines, instability of foreign genes, etc. , to enhance the affinity, prolong the half-life in the body, and enhance the efficacy

Active Publication Date: 2018-06-01
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are many GLP-1 analogs on the market for the treatment of diabetes, but due to the degradation of dipeptidyl peptidase IV and the rapid clearance of the blood, the half-life of the drug is relatively short, and the efficacy cannot be maintained for a long time. A commonly used strategy is amino acid mutation and Fc fusion of GLP-1
In addition, the DHFR expression system is generally used in the industry at present, but the continuous pressurization of MTX causes the instability of the exogenous gene in the cell genome, which not only results in the lack of stability of the cell line, but also a low expression level. The current state of the art GLP-1 fusion The protein expression level

Method used

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  • Recombinant GLP-1 analogue Fc fusion protein optimized gene and application thereof
  • Recombinant GLP-1 analogue Fc fusion protein optimized gene and application thereof
  • Recombinant GLP-1 analogue Fc fusion protein optimized gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of recombinant expression plasmid pXC17.4-rGLP-1-Fc

[0029] 1. Full gene synthesis of recombinant GLP-1 analogue Fc fusion protein gene (rGLP-1-Fc)

[0030] According to the literature (Haryadi R, Ho S, Kok YJ, et al. Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells[J]. Plos One, 2015, 10(2): e0116878.) Through screening and optimization, the signal peptide A (SEQ ID NO:1, 19aa) was obtained, and according to the patent (A·M·Vick, R·L·Xiaomilican, W·Gleissner.GLP -1 analogue fusion protein, CN 1802386B[P].2010.) published the amino acid sequence of GLP-1 analogue (total 275aa), combining the two amino acid sequences, according to the Chinese hamster ovary cell (CHO cell) Optimize the strategy, optimize the sequence, add HindIII and Kozak sequences to the 5'end of the sequence, add double stop codons and EcoRI to the 3'end of the sequence, and obtain the GLP-1 analogue Fc fusi...

Embodiment 2

[0033] Example 2 Stable Cell Pool Screening

[0034] 1. pXC17.4-rGLP-1-Fc electroporation

[0035] Use the PvuI restriction site to linearize the plasmid pXC17.4-rGLP-1-Fc, and after purification, filter sterilization and sterility testing, the linearized plasmid pXC17.4-rGLP-1-Fc (concentration 0.4 ug / uL). Also prepare 2×10 7 The exponential growth phase CHOK1SV GS-KO cells are added to two shock cups, each electrode cup contains 1×10 7 cells, the volume is 0.7mL, each add 100μL of 0.4 ug / uL linearized DNA into the electroporation cup, and linearize plasmid pXC17 according to the preset program (pulse 300V, 900μF, resistance ∞, exponential wave, 4mm gap). 4-rGLP-1-Fc was electrotransfected into CHOK1SV GS-KO recipient cells.

[0036] 2. The transfected cells are spread on 96-well plates and pool screening

[0037] Resuspend the cells after the electric shock in a 1L conical flask containing 200mL CD CHO AGT medium, mix gently, pour into the sample tank, use a drain gun to draw the ...

Embodiment 3

[0040] Example 3 Monoclonal ClonePix2 screening

[0041] 1.Pool semi-solid medium

[0042] 1)Pool semi-solid medium culture formula

[0043] Table 1 The formula of semi-solid medium for cell Pool cloning

[0044]

[0045] Blow and suck evenly at room temperature and wait for the bubbles to disappear slowly.

[0046] 2)Pool semi-solid medium

[0047] According to the 150cells / 2mL semi-solid medium per well after plating, add the required amount of cell liquid to the semi-solid medium. Use a pipette to gently mix the semi-solid medium with the cells, and let it stand at room temperature for a few minutes to make the large bubbles disappear or break. Use a pipette to suck up 2mL of the above mixed cell liquid and slowly add it to each hole in the six-well plate. Be careful not to mix in air bubbles. If there are air bubbles, just pick them up gently. After aliquoting and spreading, cover the lid and place at 37℃, 5% CO 2 Stand still and cultivate.

[0048] 2. ClonePix2 selects a single cl...

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PUM

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Abstract

The invention belongs to the field of bio-pharmacy, and particularly relates to a recombinant GLP-1 analogue Fc fusion protein optimized gene (rGLP-1-Fc for short) and an application thereof. The human GLP-1 analogue Fc fusion gene is optimized, introduced into a plasmid and transfected into CHOK1SV GS-KO cells, and after screening, a positive CHOK1SV GS-KO cell line expressing GLP-1 analog Fc fusion protein stably and efficiently is obtained. The constructed optimized gene has the advantages that the secretion and expression amount in positive cell strains is increased significantly, the secreted fusion protein has higher biological activity and can directly act on a GLP-1 receptor, and affinity is higher; blood sugar and glycosylated hemoglobin can be reduced rapidly, efficiently and permanently; the optimized gene has functions of improving islet beta cell function, reducing blood fat, reducing weight, delaying gastric emptying, increasing satiety and the like.

Description

Technical field [0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to an optimized gene of a recombinant GLP-1 analogue Fc fusion protein (referred to as rGLP-1-Fc) and its application. Background technique [0002] According to the latest IDF data, the number of diabetes patients worldwide rose to 415 million in 2015, and the incidence of diabetes was 8.8%. It is estimated that by 2040 there will be 642 million diabetes patients worldwide, and the incidence will rise to 10.4%. In 2015, the number of diabetes patients (20-79 years old) in China increased by 11.2 million compared with 2013, reaching 109.6 million, ranking first in the world. It is estimated that by 2040, the number of diabetic patients aged 20-79 will reach 150.7 million. In most countries, diabetes and its complications are the main cause of premature death, and 50% or more of the deaths of diabetic patients are attributed to cardiovascular disease. In 2015, the number of p...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/85C12N5/10
CPCC07K14/605C07K2319/02C07K2319/30C12N5/0682C12N15/625C12N15/85C12N2510/02C12N2800/107
Inventor 肖海鹏陈英张奕敏张玮龚庆伟张晓焰马利陈小锋李文佳
Owner SUNSHINE LAKE PHARM CO LTD
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