Recombinant variant FGF21 protein as well as preparation method and application thereof

A protein and variant technology, applied in the field of biomedicine, can solve the problems of not significantly improving blood sugar, etc., and achieve the effect of easy linear method, high yield, and reduced production cost

Active Publication Date: 2021-11-30
赣江中药创新中心
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, clinical trials found that FGF21 did not significantly improve blood sugar in diabetic patients, which may be related to the biological activity and in vivo stability of the designed FGF21 mutants, so genetic engineering and chemical modification of FGF21 have gradually become a research topic in recent years. Hotspots

Method used

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  • Recombinant variant FGF21 protein as well as preparation method and application thereof
  • Recombinant variant FGF21 protein as well as preparation method and application thereof
  • Recombinant variant FGF21 protein as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0031] Expression and purification of FGF21 protein

[0032] (1) Construction of expression vector

[0033] The designed mFGF21 gene (see SEQ ID NO: 1 in the sequence table) and the wild-type hFGF21 gene (GenBank: PA260867.1) were synthesized by Nanjing GenScript, and Nde I and Hind III were added to both ends of the target gene. Restriction sites. The synthetic target genes containing restriction sites were ligated into pET30a(+) vectors, and then transformed into Escherichia coli DH5α. Positive clones were picked, identified by enzyme digestion and sequenced to construct successful recombinant expression vectors pET30a-mFGF21 and pET30a-hFGF21.

[0034] (2) Expression identification of FGF21 protein

[0035]The recombinant expression vectors pET30a-mFGF21 and pET30a-hFGF21 were respectively transformed into BL21(DE3) competent cells. Single colonies were picked and inoculated into 20 mL of LB medium containing Kan (50 μg / mL), cultured at 37 °C for 12 h, and inoculated in...

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Abstract

The invention discloses a recombinant human fibroblast growth factor 21 analogue (mFGF21) and a preparation method thereof. The method comprises the following steps: connecting a recombinant mFGF21 protein coding gene with an expression vector to obtain a recombinant plasmid, transforming host cells, screening positive stable expression engineering bacteria, performing high-density fermentation, centrifugally collecting thalli, performing high-pressure homogenate crushing, performing inclusion body enrichment, performing washing, performing denaturation, performing renaturation and performing buffer solution replacement to obtain the recombinant mFGF21 protein; and by adopting ion-exchange chromatography, performing fine purification and post-treatment of a membrane separation technology to obtain the mFGF21 protein with electrophoresis and liquid phase purity greater than 98%. The invention also discloses an application of the recombinant mFGF21 protein to treatment of metabolic diseases such as obesity, diabetes, hyperlipidemia and non-alcoholic fatty liver diseases. Compared with wild type hFGF21, the mFGF21 protein disclosed by the invention has the advantages that the stability and the biological activity are obviously improved, and the mFGF21 protein has a more obvious effect on improving the blood sugar and blood fat level and fatty liver of a model animal.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a recombinant variant FGF21 protein and its preparation method and application. Background technique [0002] With the continuous improvement of living standards, metabolic diseases represented by diabetes mellitus (DM) and non-alcoholic fatty liver disease (NASH) have become invisible killers threatening human health. There are more than 100 million insulin-resistant diabetic patients (T2DM) in my country, and complications such as blindness, neuropathy, hypertension, liver and kidney disease, and cardiovascular disease will occur in the later stage of the disease. The global market will reach 120 billion US dollars in 2025. Fatty liver has become a major health threat that has been seriously underestimated, and the number of patients has exceeded that of diabetes (T2DM 100 million). Fatty liver may not only cause serious liver lesions or even cancer, but also may cause card...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/50C12N15/12C12N15/70C12N1/21C07K1/36C07K1/34C07K1/18A61K38/18A61P3/10A61P3/04A61P3/06A61P1/16A61P9/10A61P35/00C12R1/19
CPCC07K14/50C12N15/70A61P3/10A61P3/04A61P3/06A61P1/16A61P9/10A61P35/00A61K38/00
Inventor 梁鑫淼叶贤龙郭志谋胡飞
Owner 赣江中药创新中心
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