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Recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases

A technology of human fibroblasts and fusion proteins, 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 good blood sugar fluctuations and good hypoglycemic effects

Inactive Publication Date: 2017-02-15
HENAN NORMAL UNIV
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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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  • Recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases
  • Recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases
  • Recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases

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Experimental program
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Effect test

Embodiment 1

[0026] Construction of hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 expression vectors

[0027] According to the codon preference of Escherichia coli, 4 genes were designed, and their nucleotide sequences are shown in the sequence listing as SEQ ID NO:1 (hFGF21-HSA), SEQ ID NO:2 (HSA-hFGF21), SEQ ID NO:3 (hFGF21-3DHSA) and SEQ ID NO: 4 (3DHSA-hFGF21). These four 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.

[0028] The four synthetic vectors and pET30a(+) containing their respective target gene fragments were double-enzyme-digested with NdeI and Bam HI respectively. Using T4 DNA ligase, the four target fragments were respectively ligated with the prokaryotic expression vector pET30a(+), the ligation reaction system was 10 µL, mixed well, ligated overnight at 4°C, and then transformed into E. coli DH5α respectively. The positive clones were picked and identified by...

Embodiment 2

[0030] Expression and purification of four fusion proteins hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21

[0031] (1) Transformation, culture and induction of expression

[0032] The four recombinant plasmids pET30a-hFGF21-HSA, pET30a-HSA-hFGF21, pET30a-hFGF21-3DHSA and pET30a-3DHSA-hFGF21 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 600 At 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 centrifu...

Embodiment 3

[0037] In vivo half-life detection of four proteins hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21

[0038]Eighteen rabbits weighing about 2 kg were selected and randomly divided into 6 groups. Each group was subcutaneously injected with 6 kinds of proteins hFGF21, hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21, the dose was 30nmol / kg, at 0h, 1h, 3h, 5h, 7h, 24h after administration, About 800 μL of blood was collected from the ear vein. Centrifuge at 12000r / m for 10min, take the supernatant and store it at -20°C for later use.

[0039] The in vivo half-life of six proteins was determined by ELISA indirect method: hFGF21, hFGF21-HSA, HSA-hFGF21, hFGF21-3DHSA and 3DHSA-hFGF21 proteins (2μg / mL, 0.2μg / mL, 200ng / mL, 20ng / mL and 2ng / mL) to establish the standard curve of protein concentration respectively, the diluted standard protein and serum were coated on the microtiter plate, and the content of the target protein in each serum was determined by ELISA indirect ...

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Abstract

The invention discloses recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases. A nucleotide sequence of coding genes of the recombinant human fibroblast growth factor 21 fusion protein and an expression vector are connected to obtain a recombinant expression vector; the recombinant expression vector converts host cells, high-expression positive host cells are screened out, the cells are cultured, the recombinant human fibroblast growth factor 21 fusion protein is induced to be expressed, thalli are collected, breaking, centrifuging, clarifying and purifying are carried out, and the target product recombinant human fibroblast growth factor 21 fusion protein is obtained. The invention further discloses the application of the recombinant human fibroblast growth factor 21 fusion protein in preparation of the medicine for treating metabolic diseases. The recombinant human fibroblast growth factor 21 fusion protein can well control blood sugar fluctuation and remarkably improve the level of glycosylated hemoglobin and triglyceride.

Description

technical field [0001] The present invention relates to the field of recombinant proteins, in particular to a recombinant human fibroblast growth factor 21 fusion protein and a preparation method thereof, and the present invention also relates to the use of the recombinant human fibroblast growth factor 21 fusion protein in the preparation of drugs for treating metabolic diseases , belongs to the technical field of fibroblast growth factor. 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 di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K38/18A61K47/64A61P3/10A61P3/04A61P3/00
CPCC07K14/50A61K38/00C07K2319/31
Inventor 叶贤龙齐剑英吴云舟朱升龙
Owner HENAN NORMAL UNIV
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