Mutant fibroblast growth factor and use thereof in treating endocrine diseases
A technology of fibroblasts and growth factors, applied in the direction of fibroblast growth factors, growth factors/inducing factors, medical preparations containing active ingredients, etc., can solve the problems of wasting manpower and material resources, and achieve lower blood sugar levels, medication long-lasting effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0025] Example 1 Cloning of FGF-21 Gene
[0026] According to the nucleotide sequence of the FGF-21 gene with the signal peptide removed, primers were designed, and the FGF-21 gene with the signal peptide removed was obtained by complementary extension. Primers for FGF-21 mutants were designed using the online software Primer5.0.
[0027] Using extracted human liver total RNA as a template, Oligo(dT) 15 As primers, the first strand of cDNA was synthesized according to the instructions of M-MLV reverse transcriptase M-MLVRT. The reaction system and specific operations were as follows:
[0028]
[0029] Water bath at 70°C for 5 minutes, place on ice for 5 minutes, add in sequence:
[0030]
[0031] 37 ℃ water bath for 2 h, 70 ℃ water bath for 15 min, take 2 μl for PCR amplification reaction. The mature polypeptide cDNA of FGF-21 mutant was amplified by conventional PCR method, and the PCR reaction (50 μl system) was as follows:
[0032]
[0033] P1: 5' GGTCTCTAGGT ...
Embodiment 2
[0035] Example 2 Preparation and Activity Detection of Mutant Fibroblast Growth Factor-21
[0036] 1. Construction of mutant FGF-21 gene expression vector
[0037] Ligate the target fragment of the FGF-21 mutant recovered in Example 1 with the prokaryotic expression vector pET30a (+), and the ligation reaction system (10 μl) is as follows:
[0038]
[0039]A total of 10 μl of the system was mixed, and ligated at 4°C overnight. After enzyme digestion and identification, the recombinant plasmid pET-30a-FGF-21 was constructed;
[0040] 2. Obtaining of mutant FGF-21 protein
[0041] (1), induced expression
[0042] Transform the pET30a plasmid containing the correct sequence into FGF-21 and the SUMO tag into the expression strain Transetta (DE3) (Beijing Quanshijin Biotechnology Co., Ltd., catalog number: CD801). Transformed single colonies were inoculated into 5 mL of LB medium, cultured at 37°C for 10 h, inoculated in 500 mL of LB medium containing penicillin (50 mg / mL) a...
Embodiment 3
[0053] Example 3 In vivo activity test of mutant FGF-21 protein of the present invention
[0054] Spontaneous type 2 diabetes db / db model mice (Shanghai Slack Experimental Animal Co., Ltd., Animal Quality Certificate No. SCXK (Shanghai)) were randomly divided into 3 groups, 5 mice in each group. By subcutaneous administration, the negative control group was injected with normal saline; the wild group and the mutant group were injected with protein at a dose of 0.75 mg / kg. The experiment started at 8:00 in the morning (at this time, the blood glucose of the model animals was the highest value in a day), and they were free to eat and drink during the experiment. Detect the blood sugar changes of the model animals after different treatments, and the experimental data obtained are statistically analyzed. The results are as follows: image 3 shown.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More