Mutant fibroblast growth factor and use thereof in treating endocrine diseases
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A technology for fibroblasts and endocrine diseases, applied in the directions of fibroblast growth factors, growth factors/inducing factors, metabolic diseases, etc. With the effect of development
Active Publication Date: 2012-07-25
TIANJIN TASLY PHARMA CO LTD
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In previous biopharmaceutical research, random mutations were often used for protein modification. Due to the randomness of mutations and the complexity of protein functions, random mutations are often counterproductive and waste a lot of manpower and material resources.
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Embodiment 1
[0024] Example 1 Cloning of FGF-21 Gene
[0025] 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.
[0026] 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:
[0027]
[0028] Water bath at 70°C for 5 minutes, place on ice for 5 minutes, add in sequence:
[0029]
[0030] 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:
[0031]
[0032] P1: 5' GGTCT...
Embodiment 2
[0036] Example 2 Preparation and Activity Detection of Mutant Fibroblast Growth Factor-21
[0037] 1. Construction of mutant FGF-21 gene expression vector
[0038] 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:
[0039]
[0040] 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;
[0041] 2. Obtaining of mutant FGF-21 protein
[0042] (1), induced expression
[0043] Transform the recombinant plasmid pET-30a-FGF-21 containing the correct sequence into the expression strain Trans etta (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) at ...
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Abstract
The invention discloses a mutant fibroblast growth factor (FGF-21) and use of the mutant fibroblast growth factor in treating endocrine diseases. The amino acid sequence of the fibroblast growth factor-21 disclosed by the invention is shown as SEQ ID NO: 2, and the sequence of a gene for encoding the mutant fibroblast growth factor is shown as SEQ ID NO: 1. According to the invention, a wild typeFGF-21 is used as a template, two arginine (Arg) residues are introduced through a downstream primer, and a mutant is obtained through polymerase chain reaction (PCR). The strong basicity and positive charges in the physiological condition of Arg are mainly utilized so that the isoelectric point of FGF-21 is up-regulated, and FGF-21 binding to the surfaces of cell membranes is facilitated. The results of an animal experiment show that the mutant FGF-21 disclosed by the invention can more effectively reduce the blood glucose level in an animal, and furthermore, the mutant disclosed by the invention has the advantages of fast onset of drug action, lasting drug effect and the like in reducing the blood glucose level. The mutant GF-21 disclosed by the invention can be used as a medicine to treat endocrine diseases such as diabetes, metabolic syndrome, lipid metabolism disorder and the like.
Description
technical field [0001] The present invention relates to fibroblast growth factor, in particular to a mutated fibroblast growth factor and a preparation method thereof. The present invention also relates to the use of the mutated fibroblast growth factor in the preparation of medicines for treating endocrine diseases, which belongs to the field of fibroblast growth factor field. [0002] Background technique [0003] Diabetes is the third biggest killer of humans after cardiovascular and cerebrovascular diseases and tumors. With changes in lifestyle and diet, the prevalence of diabetes worldwide is on the rise. It is estimated that the number of people with diabetes in the world will increase to 366 million by 2030, and diabetes will become a heavy burden for every country in the world. [0004] For type 2 diabetes, the vast majority of patients will inevitably be treated with drugs. Among them, there are three major categories of oral drugs: the first category is yellow...
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