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

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...

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 is around 1g / L or lower than 1g / L. For example, the GLP-1-IgG2σFc fusion protein sequence constructed by Yang Yi et al. L(Yang Yi, Wan Deyou, Liu Yunhui, et al. Eukaryotic expression and biological activity identification of long-acting GLP-1-IgG2σFc fusion protein[J]. Biotechnology Communications, 2016,27(2):173-177.) ; In the patent (Xu Bixiong, Guo Qiran, Feng Jinmeng. A recombinant human GLP-1-Fc fusion protein:, CN 104327187A[P].2015.), the expression level of GLP-1-Fc fusion protein is also about 1g / L ; In the patent (Huang Yanshan, Yang Zhiyu, Xu Zhengxue, etc. GLP-1 analog fusion protein and its preparation method and use:, CN103408669B[P].2016.), the yield of GLP-1-Fc fusion protein is only 10μg / mL

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