Human fibroblast growth factor 21 recombinant protein as well as preparation method and application thereof

A technology of human fibroblasts and growth factors, applied in the fields of fibroblast growth factors and recombinant proteins, can solve the problems of increasing the difficulty of purification, the risk of immune reactions, etc., and achieve the effects of good blood sugar fluctuations, increased activity, and lower blood sugar levels

Active Publication Date: 2016-12-14
无锡代达康健生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the expression and storage of albumin fusion protein are accompanied by degradation and aggregation, which increases the difficulty of purification and the risk of immune reaction during clinical administration (Yao, X.Q., et al., Degradation of HSA-AX15(R13K) when expressed inPichia pastoris can be reduced via the disruption of YPS1 gene in this yeast.J Biotechnol, 2009. 139(2): p. 131-6. Cordes, A.A., et al., Selective domainstabilization as a strategy to reduce fusion protein aggregation. J PharmSci, 2012. 101(4): p. 1400-9. Cordes, A.A., J.F. Carpenter, and T.W.Randolph, Selective domain stabilization as a strategy to reduce human serum albumin-human granulocyte colony stimulating factor Pharma aggregation Srateci J , 2012. 101(6): p. 2009-2016.)

Method used

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  • Human fibroblast growth factor 21 recombinant protein as well as preparation method and application thereof
  • Human fibroblast growth factor 21 recombinant protein as well as preparation method and application thereof
  • Human fibroblast growth factor 21 recombinant protein as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] Construction of mFGF21, mFGF21-HSA, HSA-mFGF21, mFGF21-3DHSA and 3DHSA-mFGF21 expression vectors

[0030] According to the codon preference of Escherichia coli, five genes were designed, the nucleotide sequences of which are shown in the sequence listing as SEQ ID NO: 1 (mFGF21), SEQ ID NO: 2 (mFGF21-HSA), SEQ ID NO: 3 (HSA -mFGF21), SEQ ID NO:4 (mFGF21-3DHSA) and SEQ ID NO:5 (3DHSA-mFGF21). These five genes were sent to Shanghai Jierui Biological Co., Ltd. for synthesis, and NdeI and BamHI restriction sites were designed at both ends of each gene.

[0031] The 5 synthetic vectors and pET30a(+) containing the respective target gene fragments were double-digested with NdeI and Bam HI respectively. After the enzyme digestion was completed, the respective target fragments were recovered from the gel. Using T4 DNA ligase, the five target fragments were respectively connected to the prokaryotic expression vector pET30a (+), the ligation reaction system was 10 µL, mixed well...

Embodiment 2

[0033] Expression and purification of mFGF21 protein

[0034] (1) Transformation, culture and induction of expression

[0035] The recombinant plasmid pET30a-mFGF21 containing the correct sequence was transformed into the expression strain Rosseta (DE3) (Beijing Quanshijin Biotechnology Co., Ltd., catalog number: CD801). Transformed single colonies were inoculated into 20 mL of LB medium containing Kan (50 µg / mL), cultured at 37 °C for 8 h, and inoculated into another 20 mL of LB medium containing Kan (50 µg / mL) at a volume ratio of 1:100 Medium, cultured at 37°C, when A 600 At about 0.35, add IPTG to a final concentration of 0.25mmol / L for induction. The induction temperature is 30°C. Harvest the bacteria after 5 hours and resuspend the bacteria in Lysis buffer (20mmol / L Tris, 150mmol / L NaCl, pH 8.0) The cells were crushed and centrifuged, and the supernatant and precipitate were collected for 12wt% SDS-PAGE electrophoresis analysis. Such as figure 1 As shown, lane 1: pro...

Embodiment 3

[0040] Expression and purification of four fusion proteins mFGF21-HSA, HSA-mFGF21, mFGF21-3DHSA and 3DHSA-mFGF21

[0041] (1) Transformation, culture and induction of expression

[0042] The four recombinant plasmids pET30a-mFGF21-HSA, pET30a-HSA-mFGF21, pET30a-mFGF21-3DHSA and pET30a-3DHSA-mFGF21 containing the correct sequence were respectively transformed into the expression strain Rosseta (DE3) (Beijing Quanshijin Biotechnology Co., Ltd. , catalog number: CD801). Transformed single colonies were inoculated into 20 mL of LB medium containing Kan (50 µg / mL), cultured at 37 °C for 8 h, and inoculated into another 20 mL of LB medium containing Kan (50 µg / mL) at a volume ratio of 1:100 Medium, cultured at 37°C, when A 600At about 0.35, add IPTG to a final concentration of 0.25mmol / L for induction, and the induction temperature is 30°C. Harvest the cells after 5h, and use Binding buffer (20mmol / L Na 3 PO 4 , pH 7.0) to resuspend the bacteria, break the bacteria and centrifug...

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Abstract

The invention discloses a recombinant protein of a human fibroblast growth factor 21 as well as a preparation method and application thereof. The preparation method comprises the following steps: linking a nucleotide sequence of a coding gene of the human fibroblast growth factor 21 recombinant protein with an expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into host cells, screening high-expression positive host cells, culturing the cells, inducing the cells to express the human fibroblast growth factor 21 recombinant protein, collecting thalli, breaking, and carrying out centrifuging, clarification and purification, so as to obtain the target product, namely the human fibroblast growth factor 21 recombinant protein. The invention further discloses application of the human fibroblast growth factor 21 recombinant protein in the preparation of drugs for treating metabolic diseases. Compared with a wild hFGF21 protein, the human fibroblast growth factor 21 recombinant protein (mFGF21) has the advantages that the activity is remarkably improved, and the blood glucose level in a diabetic mouse can be relatively effectively reduced.

Description

technical field [0001] The present invention relates to the field of recombinant proteins, in particular to a recombinant protein of human fibroblast growth factor 21 and a preparation method thereof. The present invention also relates to the use of the recombinant protein of human fibroblast growth factor 21 in the preparation of drugs for treating metabolic diseases, which belongs to Fibroblast Growth Factor Technical Field. Background technique [0002] Diabetes mellitus (DM) is a chronic metabolic disease caused by pancreatic dysfunction, characterized by hyperglycemia, and is a lifelong disease. Its pathogenesis is caused by insulin resistance or decreased insulin secretion, which leads to the disorder of glucose and lipid metabolism in the body. With the aging of the world population, diabetes has become a common and frequently-occurring disease, and has become the third most life-threatening disease after cancer and cardiovascular and cerebrovascular diseases (Nathan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/50C12N15/12C12N15/70C12N1/21A61K38/18A61P3/10A61P3/04A61P3/00
Inventor 叶贤龙齐剑英仉晓文
Owner 无锡代达康健生物医药科技有限公司
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